Gastrin releasing peptide compounds

ABSTRACT

New and improved compounds for use in radiodiagnostic imaging or radiotherapy having the formula M-N—O—P-G, wherein M is the metal chelator (in the form complexed with a metal radionuclide or not), N—O—P is the linker, and G is the GRP receptor targeting peptide. Methods for imaging a patient and/or providing radiotherapy to a patient using the compounds of the invention are also provided. A method for preparing a diagnostic imaging agent from the compound is further provided. A method for preparing a radiotherapeutic agent is further provided.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/341,577, filed Jan. 13, 2003, the contents of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to novel radionuclide-labeled gastrin releasingpeptide (GRP) compounds which are useful as diagnostic imaging agents orradiotherapeutic agents. These GRP compounds include the use of novellinkers between a metal chelator and the targeting peptide, whichprovides for improved pharmacokinetics.

BACKGROUND OF THE INVENTION

The use of radiopharmaceuticals (e.g., diagnostic imaging agents,radiotherapeutic agents) to detect and treat cancer is well known. Inmore recent years, the discovery of site-directed radiopharmaceuticalsfor cancer detection and/or treatment has gained popularity andcontinues to grow as the medical profession better appreciates thespecificity, efficacy and utility of such compounds.

These newer radiopharmaceutical agents typically consist of a targetingagent connected to a metal chelator, which can be chelated to (e.g.,complexed with) a diagnostic metal radionuclide such as, for example,technetium or indium, or a therapeutic metal radionuclide such as, forexample, lutetium, yttrium, or rhenium. The role of the metal chelatoris to hold (i.e., chelate) the metal radionuclide as theradiopharmaceutical agent is delivered to the desired site. A metalchelator which does not bind strongly to the metal radionuclide wouldrender the radiopharmaceutical agent ineffective for its desired usesince the metal radionuclide would therefore not reach its desired site.Thus, further research and development led to the discovery of metalchelators, such as that reported in U.S. Pat. No. 5,662,885 to Pollaket. al., hereby incorporated by reference, which exhibited strongbinding affinity for metal radionuclides and the ability to conjugatewith the targeting agent. Subsequently, the concept of using a “spacer”to create a physical separation between the metal chelator and thetargeting agent was further introduced, for example in U.S. Pat. No.5,976,495 to Pollak et. al., hereby incorporated by reference.

The role of the targeting agent, by virtue of its affinity for certainbinding sites, is to direct the radiopharmaceutical agent containing themetal radionuclide to the desired site for detection or treatment.Typically, the targeting agent may include a protein, a peptide, orother macromolecule which exhibits a specific affinity for a givenreceptor. Other known targeting agents include monoclonal antibodies(MAbs), antibody fragments (F_(ab)'s and (F_(ab))₂'s), and receptor-avidpeptides. Donald J. Buchsbaum, “Cancer Therapy with RadiolabeledAntibodies; Pharmacokinetics of Antibodies and Their Radiolabels;Experimental Radioimmunotherapy and Methods to Increase TherapeuticEfficacy,” CRC Press, Boca Raton, Chapter 10, pp. 115-140, (1995);Fischman, et al. “A Ticket to Ride: Peptide Radiopharmaceuticals,” TheJournal of Nuclear Medicine, vol. 34, No. 12, (December 1993).

In recent years, it has been learned that some cancer cells containgastrin releasing peptide (GRP) receptors (GRP-R) of which there are anumber of subtypes. In particular, it has been shown that several typesof cancer cells have over-expressed or uniquely expressed GRP receptors.For this reason, much research and study have been done on GRP and GRPanalogues which bind to the GRP receptor family. One such analogue isbombesin (BBN), a 14 amino acid peptide (i.e., tetradecapeptide)isolated from frog skin which is an analogue of human GRP and whichbinds to GRP receptors with high specificity and with an affinitysimilar to GRP.

Bombesin and GRP analogues may take the form of agonists or antagonists.Binding of GRP or BBN agonists to the GRP receptor increases the rate ofcell division of these cancer cells and such agonists are internalizedby the cell, while binding of GRP or BBN antagonists generally does notresult in either internalization by the cell or increased rates of celldivision. Such antagonists are designed to competitively inhibitendogenous GRP binding to GRP receptors and reduce the rate of cancercell proliferation. See, e.g., Hoffken, K.; Peptides in Oncology II,Somatostatin Analogues and Bombesin Antagonists (1993), pp. 87-112. Forthis reason, a great deal of work has been, and is being pursued todevelop BBN or GRP analogues that are antagonists. E.g., Davis et al.,Metabolic Stability and Tumor Inhibition of Bombesin/GRP ReceptorAntagonists, Peptides, vol. 13, pp. 401-407, 1992.

In designing an effective radiopharmaceutical compound for use as adiagnostic or therapeutic agent for cancer, it is important that thedrug have appropriate in vivo targeting and pharmacokinetic properties.For example, it is preferable that the radiolabeled peptide have highspecific uptake by the cancer cells (e.g., via GRP receptors). Inaddition, it is also preferred that once the radionuclide localizes at acancer site, it remains there for a desired amount of time to deliver ahighly localized radiation dose to the site.

Moreover, developing radiolabeled peptides that are cleared efficientlyfrom normal tissues is also an important factor for radiopharmaceuticalagents. When biomolecules (e.g., MAb, F_(ab) or peptides) labeled withmetallic radionuclides (via a chelate conjugation), are administered toan animal such as a human, a large percentage of the metallicradionuclide (in some chemical form) can become “trapped” in either thekidney or liver parenchyma (i.e., is not excreted into the urine orbile). Duncan et al.; Indium-111-DiethylenetriaminepentaaceticAcid-Octreotide Is Delivered in Vivo to Pancreatic, Tumor Cell, Renal,and Hepatocyte Lysosomes, Cancer Research 57, pp. 659-671, (Feb. 15,1997). For the smaller radiolabeled biomolecules (i.e., peptides orF_(ab)), the major route of clearance of activity is through the kidneyswhich can also retain high levels of the radioactive metal (i.e.,normally >10-15% of the injected dose). Retention of metal radionuclidesin the kidney or liver is clearly undesirable. Conversely, clearance ofthe radiopharmaceutical from the blood stream too quickly by the kidneyis also undesirable if longer diagnostic imaging or high tumor uptakefor radiotherapy is needed.

Subsequent work, such as that in U.S. Pat. No. 6,200,546 and US2002/0054855 to Hoffman, et. al, hereby incorporated by reference, hasattempted to overcome this problem by forming a compound having thegeneral formula X—Y—B wherein X is a group capable of complexing ametal, Y is a covalent bond on a spacer group and B is a bombesinagonist binding moiety. Such compounds were reported to have highbinding affinities to GRP receptors, and the radioactivity was retainedinside of the cells for extended time periods. In addition, in vivostudies in normal mice have shown that retention of the radioactivemetal in the kidneys was lower than that known in the art, with themajority of the radioactivity excreted into the urine.

New and improved radiopharmaceutical compounds which have improvedpharmacokinetics and improved kidney excretion (i.e., lower retention ofthe radioactive metal in the kidney) have now been found for diagnosticimaging and therapeutic uses. For diagnostic imaging, rapid renalexcretion and low retained levels of radioactivity are critical forimproved images. For radiotherapeutic use, slower blood clearance toallow for higher tumor uptake and better tumor targeting with low kidneyretention are critical.

SUMMARY OF THE INVENTION

In an embodiment of the present invention, there is provided new andimproved compounds for use in radiodiagnostic imaging or radiotherapy.The compounds include a chemical moiety capable of complexing amedically useful metal ion or radionuclide (metal chelator) attached toa GRP receptor targeting peptide by a linker or spacer group.

In general, compounds of the present invention may have the formula:M-N—O—P-G

wherein M is the metal chelator (in the form complexed with a metalradionuclide or not), N—O—P is the linker, and G is the GRP receptortargeting peptide.

The metal chelator M may be any of the metal chelators known in the artfor complexing with a medically useful metal ion or radionuclide.Preferred chelators include DTPA, DOTA, DO3A, HP-DO3A, EDTA, TETA, EHPG,HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM, or peptidechelators, such as, for example, those discussed herein. The metalchelator may or may not be complexed with a metal radionuclide, and mayinclude an optional spacer such as a single amino acid. Preferred metalradionuclides for scintigraphy or radiotherapy include ⁹⁹Tc, ⁵¹Cr, ⁶⁷Ga,⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y,¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³ Ru, ¹⁸⁶Re,¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ^(117m)Sn,¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metal will bedetermined based on the desired therapeutic or diagnostic application.For example, for diagnostic purposes the preferred radionuclides include⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In, with ^(99m)Tc, and ¹¹¹In beingparticularly preferred. For therapeutic purposes, the preferredradionuclides include ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Luand ⁹⁰Y being particularly preferred. A most preferred chelator used incompounds of the invention is 1-substituted 4,7,10-tricarboxymethyl1,4,7,10 tetraazacyclododecane triacetic acid (DO3A).

In one embodiment, the linker N—O—P contains at least one non-alphaamino acid.

In another embodiment, the linker N—O—P contains at least onesubstituted bile acid.

In yet another embodiment, the linker N—O—P contains at least onenon-alpha amino acid with a cyclic group.

The GRP receptor targeting peptide may be GRP, bombesin or anyderivatives or analogues thereof. In a preferred embodiment, the GRPreceptor targeting peptide is a GRP or bombesin analogue which acts asan agonist. In a particularly preferred embodiment, the GRP receptortargeting peptide is a bombesin agonist binding moiety disclosed in U.S.Pat. No. 6,200,546 and US 2002/0054855, incorporated herein byreference.

There is also provided a novel method of imaging using the compounds ofthe present invention.

There is further provided a novel method for preparing a diagnosticimaging agent comprising the step of adding to an injectable imagingmedium a substance containing the compounds of the present invention.

A novel method of radiotherapy using the compounds of the invention isalso provided, as is a novel method for preparing a radiotherapeuticagent comprising the step of adding to an injectable therapeutic mediuma substance comprising a compound of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical representation of a series of chemical reactionsfor the synthesis of intermediate C((3β,5β)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid), from A(Methyl-(3β,5β)-3-aminocholan-24-ate) and B((3β,5β)-3-aminocholan-24-oic acid), as described in Example I;

FIG. 1B is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L62), as described in Example I;

FIG. 2A is a graphical representation of the sequential reaction for thesynthesis ofN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L70), as described in Example II;

FIG. 2B is a general graphical representation of the sequential reactionfor the synthesis ofN-[4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L73),N-[3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L15), andN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L16), as described in Example II;

FIG. 2C is a chemical structure of the linker used in the synthesisreaction of FIG. 2B for synthesis ofN-[4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L73), as described in Example II;

FIG. 2D is a chemical structure of the linker used in the synthesisreaction of FIG. 2B for synthesis ofN-[3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L115), as described in Example II;

FIG. 2E is a chemical structure of the linker used in the synthesisreaction of FIG. 2B for synthesis ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L116), as described in Example II;

FIG. 2F is a graphical representation of the sequential reaction for thesynthesis ofN-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]glycyl-4-piperidinecarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L74), as described in Example II;

FIG. 3A is a graphical representation of a series of chemical reactionsfor the synthesis of intermediate(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oicacid (C), as described in Example III;

FIG. 3B is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L67), as described in Example III;

FIG. 3C is a chemical structure of (3β,5β)-3-Amino-12-oxocholan-24-oicacid (B), as described in Example III;

FIG. 3D is a chemical structure of(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oicacid (C), as described in Example III;

FIG. 3E is a chemical structure ofN-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L67), as described in Example III;

FIG. 4A is a graphical representation of a sequence of reactions toobtain intermediates(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic acid (3a) and(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid (3b), as described in Example IV;

FIG. 4B is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L63), as described in Example IV;

FIG. 4C is a graphical representation of the sequential reaction for thesynthesis ofN-[(3β,5β,7α,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L64), as described in Example IV;

FIG. 4D is a chemical structure of(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid (2b), asdescribed in Example IV;

FIG. 4E is a chemical structure of(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oicacid (3a), as described in Example IV;

FIG. 4F is a chemical structure of(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid (3b), as described in Example IV;

FIG. 4G is a chemical structure ofN-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L63), as described in Example IV;

FIG. 4H is a chemical structure ofN-[(3β,5β,7α,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L64), as described in Example IV;

FIG. 5A is a general graphical representation of the sequential reactionfor the synthesis of4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L71); andTrans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]cyclohexylcarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L72) as described in Example V;

FIG. 5B is a chemical structure of the linker used in compound L71 asshown in FIG. 5A and as described in Example V;

FIG. 5C is a chemical structure of the linker used in compound L72 asshown in FIG. 5B and as described in Example V;

FIG. 5D is a chemical structure of Rink amide resin functionalised withbombesin[7-14] (B), as described in Example V;

FIG. 5E is a chemical structure ofTrans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylicacid (D), as described in Example V;

FIG. 6A is a graphical representation of a sequence of reactions for thesynthesis of intermediate linker2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid (E), asdescribed in Example VI;

FIG. 6B is a graphical representation of a sequence of reactions for thesynthesis of intermediate linker4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid(H), as described in Example VI;

FIG. 6C is a graphical representation of the synthesis ofN-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L75), as described in Example VI;

FIG. 6D is a graphical representation of the synthesis ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L76), as described in Example VI;

FIG. 6E is a chemical structure of2-[(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid (C), asdescribed in Example VI;

FIG. 6F is a chemical structure of 2-(aminomethyl)benzoic acid (D), asdescribed in Example VI;

FIG. 6G is a chemical structure of2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid (E), asdescribed in Example VI;

FIG. 6H is a chemical structure of 4-(aminomethyl)-3-nitrobenzoic acid(G), as described in Example VI;

FIG. 6I is a chemical structure of4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid(H), as described in Example VI;

FIG. 6J is a chemical structure ofN-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L75), as described in Example VI;

FIG. 6K is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L76), as described in Example VI;

FIG. 7A is a graphical representation of a sequence of reactions for thesynthesis of intermediate linker[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid(E), as described in Example VII;

FIG. 7B is a graphical representation of the synthesis ofN-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-triptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L124), as described in Example VII;

FIG. 7C is a chemical structure of (4-Cyanophenoxy)acetic acid ethylester (B), as described in Example VII;

FIG. 7D is a chemical structure of (4-Cyanophenoxy)acetic acid (C), asdescribed in Example VII;

FIG. 7E is a chemical structure of[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid(E), as described in Example VII;

FIG. 7F is a chemical structure ofN-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L124), as described in Example VII;

FIG. 8A is a graphical representation of a sequence of reactions for thesynthesis of intermediate4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acid(E), as described in Example VIII;

FIG. 8B is a graphical representation of the synthesis ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L125), as described in Example VIII;

FIG. 8C is a chemical structure of 4-(Azidomethyl)-3-methoxybenzoic acidmethyl ester, (B), as described in Example VIII;

FIG. 8D is a chemical structure of 4-(Aminomethyl)-3-methoxybenzoic acidmethyl ester, (C), as described in Example VIII;

FIG. 8E is a chemical structure of4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoicacid, (E), as described in Example VIII;

FIG. 8F is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,(L125), as described in Example VIII;

FIG. 9A and FIG. 9B are graphical representations of the binding andcompetition curves described in Example X;

FIG. 10A is a graphical representation of the results of radiotherapyexperiments described in Example XXI;

FIG. 10B is a graphical representation of the results of otherradiotherapy experiments described in Example XXI;

FIG. 11 is a chemical structure ofDO3A-monoamide-Gly-Lys-(3,6,9)-trioxaundecane-1,11-dicarboxylicacid-3,7-dideoxy-3-aminocholicacid)-L-arginyl-L-glutaminyl-L-triptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L65);

FIG. 12 is a chemical structure ofN-[2-S-[[[[[12α-Hydroxy-17β-(1-methyl-3-carboxypropyl)etiocholan-3β-carbamoylmethoxyethoxyethoxyacetyl]-amino-6-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]hexanoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L66);

FIG. 13A is a chemical structure ofN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L70);

FIG. 13B is a chemical structureN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-3-carboxypropionyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L114);

FIG. 13C is a chemical structureN-[4-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]-2-hydroxy-3-propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L144);

FIG. 13D is a chemical structureN-[(3β,5β,7α,12α)-3-[[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxyethoxy]acetyl]amino]-7,12-dihydroxycholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L69); and

FIG. 13E is a chemical structureN-[4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide(L146).

FIG. 14 discloses chemical structures of intermediates which may be usedto prepare compounds L64 and L70 as described in Example XXII.

FIG. 15 is a graphical representation of the preparation of L64 usingsegment coupling as described in Example XXII.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, various aspects of the present inventionwill be further elaborated. For purposes of explanation, specificconfigurations and details are set forth in order to provide a thoroughunderstanding of the present invention. However, it will also beapparent to one skilled in the art that the present invention may bepracticed without the specific details. Furthermore, well known featuresmay be omitted or simplified in order not to obscure the presentinvention.

In an embodiment of the present invention, there is provided a new andimproved compound for use in radiodiagnostic imaging or radiotherapy.The compound includes a chemical moiety capable of complexing amedically useful metal ion or radionuclide (metal chelator) attached toa GRP receptor targeting peptide by a linker or spacer group.

In general, compounds of the present invention may have the formula:M-N—O—P-Gwherein M is the metal chelator (in the form complexed with a metalradionuclide or not), N—O—P is the linker, and G is the GRP receptortargeting peptide. Each of the metal chelator, linker, and GRP receptortargeting peptide is described in the discussion that follow.

In another embodiment of the present invention, there is provided a newand improved linker or spacer group which is capable of linking a metalchelator to a GRP receptor targeting peptide. In general, linkers of thepresent invention may have the formula:N—O—Pwherein each of N, O and P are defined throughout the specification.

Compounds meeting the criteria defined herein were discovered to haveimproved pharmacokinetic properties compared to other radiolabeled GRPreceptor targeting peptide conjugates known in the art. For example,compounds containing the linkers of the present invention were retainedin the bloodstream longer, and thus had a longer half life than priorknown compounds. The longer half life was medically beneficial becauseit permitted better tumor targeting which is useful for diagnosticimaging, and especially for therapeutic uses, where the cancerous cellsand tumors receive greater amounts of the radiolabeled peptides.

1. Metal Chelator

The term “metal chelator” refers to a molecule that forms a complex witha metal atom, wherein said complex is stable under physiologicalconditions. That is, the metal will remain complexed to the chelatorbackbone in vivo. More particularly, a metal chelator is a molecule thatcomplexes to a radionuclide metal to form a metal complex that is stableunder physiological conditions and which also has at least one reactivefunctional group for conjugation with the linker N—O—P. The metalchelator M may be any of the metal chelators known in the art forcomplexing a medically useful metal ion or radionuclide. The metalchelator may or may not be complexed with a metal radionuclide.Furthermore, the metal chelator can include an optional spacer such as asingle amino acid (e.g., Gly) which does not complex with the metal, butwhich creates a physical separation between the metal chelator and thelinker.

The metal chelators of the invention may include, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelators (see also, U.S.Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556,U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142, the disclosures ofwhich are incorporated by reference herein in their entirety), and otherchelators known in the art including, but not limited to, HYNIC, DTPA,EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see also U.S.Pat. No. 5,720,934). For example, N₄ chelators are described in U.S.Pat. Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and5,688,487, the disclosures of which are incorporated by reference hereinin their entirety. Certain N₃S chelators are described inPCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. Nos.5,662,885; 5,976,495; and 5,780,006, the disclosures of which areincorporated by reference herein in their entirety. The chelator mayalso include derivatives of the chelating ligandmercapto-acetyl-glycyl-glycyl-glycine (MAG3), which contains an N₃S, andN₂S₂ systems such as MAMA (monoamidemonoaminedithiols), DADS (N₂Sdiaminedithiols), CODADS and the like. These ligand systems and avariety of others are described in Liu and Edwards, Chem. Rev. 1999, 99,2235-2268 and references therein, the disclosures of which areincorporated by reference herein in their entirety.

The metal chelator may also include complexes containing ligand atomsthat are not donated to the metal in a tetradentate array. These includethe boronic acid adducts of technetium and rhenium dioximes, such asthose described in U.S. Pat. Nos. 5,183,653; 5,387,409; and 5,118,797,the disclosures of which are incorporated by reference herein, in theirentirety.

Examples of preferred chelators include, but are not limited to,diethylenetriamine pentaacetic acid (DTPA),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10 tetraazacyclododecanetriacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-Cl-EHPG, 5-Br-EHPG,5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, anddibenzyl-DTPA; bis-2(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED)and derivatives thereof, the class of macrocyclic compounds whichcontain at least 3 carbon atoms, more preferably at least 6, and atleast two heteroatoms (O and/or N), which macrocyclic compounds canconsist of one ring, or two or three rings joined together at the heteroring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, whereNOTA is 1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA,benzo-DOTMA, where DOTMA is1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraaceticacid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM).Examples of representative chelators and chelating groups contemplatedby the present invention are described in WO 98/18496, WO 86/06605, WO91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473,PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S. Pat. No. 5,474,756,U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6,143,274, each of which ishereby incorporated by reference in its entirety.

Particularly preferred metal chelators include those of Formula 1, 2 and3 (for ¹¹¹In and radioactive lanthanides, such as, for example ¹⁷⁷Lu,⁹⁰Y, ¹⁵³Sm, and ¹⁶⁶Ho) and those of Formula 4, 5 and 6 (for radioactive^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re) set forth below. These and other metalchelating groups are described in U.S. Pat. Nos. 6,093,382 and5,608,110, which are incorporated by reference herein in their entirety.Additionally, the chelating group of formula 3 is described in, forexample, U.S. Pat. No. 6,143,274; the chelating group of formula 5 isdescribed in, for example, U.S. Pat. Nos. 5,627,286 and 6,093,382, andthe chelating group of formula 6 is described in, for example, U.S. Pat.Nos. 5,662,885; 5,780,006; and 5,976,495, all of which are incorporatedby reference. Specific metal chelators of formula 6 includeN,N-dimethylGly-Ser-Cys; N,N-dimethylGly-Thr-Cys;N,N-diethylGly-Ser-Cys; N,N-dibenzylGly-Ser-Cys; and other variationsthereof. For example, spacers which do not actually complex with themetal radionuclide such as an extra single amino acid Gly, may beattached to these metal chelators (e.g., N,N-dimethylGly-Ser-Cys-Gly;N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly;N,N-dibenzylGly-Ser-Cys-Gly). Other useful metal chelators such as allof those disclosed in U.S. Pat. No. 6,334,996, also incorporated byreference (e.g., Dimethylgly-L-t-Butylgly-L-Cys-Gly;Dimethylgly-D-t-Butylgly-L-Cys-Gly; Dimethylgly-L-t-Butylgly-L-Cys,etc.)

Furthermore, sulfur protecting groups such as Acm (acetamidomethyl),trityl or other known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyland organothiol groups may be attached to the cysteine amino acid ofthese metal chelators.

Additionally, other useful metal chelators include:

In the above Formulas 1 and 2, R is alkyl, preferably methyl. In theabove Formula 5, X is either CH₂ or O, Y is either C₁-C₁₀ branched orunbranched alkyl; Y is aryl, aryloxy, arylamino, arylaminoacyl; Y isarylalkyl—where the alkyl group or groups attached to the aryl group areC₁-C₁₀ branched or unbranched alkyl groups, C₁-C₁₀ branched orunbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups, J is C(═O)—, OC(═O)—, SO₂—, NC(═O)—,NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—, homopolyamides orheteropolyamines derived from synthetic or naturally occurring aminoacids; all where n is 1-100. J may also be absent. Other variants ofthese structures are described, for example, in U.S. Pat. No. 6,093,382.In Formula 6, the group S—NHCOCH₃ may be replaced with SH or S-Z whereinZ is any of the known sulfur protecting groups such as those describedabove. Formula 7 illustrates one embodiment of t-butyl compounds usefulas a metal chelator. The disclosures of each of the foregoing patents,applications and references are incorporated by reference herein, intheir entirety.

In a preferred embodiment, the metal chelator includes cyclic or acyclicpolyaminocarboxylic acids such as DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DTPA(diethylenetriaminepentaacetic acid), DTPA-bismethylamide,DTPA-bismorpholineamide, DO3AN-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl,HP-DO3A, DO3A-monoamide and derivatives thereof.

Preferred metal radionuclides for scintigraphy or radiotherapy include^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸G, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb,¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru,¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd,^(117m)Sn, ¹⁴⁹ Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au and oxides or nitridesthereof. The choice of metal will be determined based on the desiredtherapeutic or diagnostic application. For example, for diagnosticpurposes (e.g., to diagnose and monitor therapeutic progress in primarytumors and metastases), the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga,⁶⁸Ga, ^(99m)Tc, and ¹¹¹In, with ^(99m)Tc and ¹¹¹In being especiallypreferred. For therapeutic purposes (e.g., to provide radiotherapy forprimary tumors and metastasis related to cancers of the prostate,breast, lung, etc.), the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Lu and ⁹⁰Y being particularlypreferred. ^(99m)Tc is particularly useful and is a preferred fordiagnostic radionuclide because of its low cost, availability, imagingproperties, and high specific activity. The nuclear and radioactiveproperties of ^(99m)Tc make this isotope an ideal scintigraphic imagingagent. This isotope has a single photon energy of 140 keV and aradioactive half-life of about 6 hours, and is readily available from a⁹⁹Mo-^(99m)Tc generator. For example, the ^(99m)Tc labeled peptide canbe used to diagnose and monitor therapeutic progress in primary tumorsand metastases. Peptides labeled with ¹⁷⁷Lu, ⁹⁰Y or other therapeuticradionuclides can be used to provide radiotherapy for primary tumors andmetastasis related to cancers of the prostate, breast, lung, etc.

2A. Linkers Containing at Least One Non-Alpha Amino Acid

In one embodiment of the invention, the linker N—O—P contains at leastone non-alpha amino acid. Thus, in this embodiment of the linker N—O—P,

-   -   N is 0 (where 0 means it is absent), an alpha or non-alpha amino        acid or other linking group;    -   O is an alpha or non-alpha amino acid; and    -   P is 0, an alpha or non-alpha amino acid or other linking group,    -   wherein at least one of N, O or P is a non-alpha amino acid.        Thus, in one example, N=Gly, 0=a non-alpha amino acid, and P=0.

Alpha amino acids are well known in the art, and include naturallyoccurring and synthetic amino acids.

Non-alpha amino acids also include those which are naturally occurringor synthetic. Preferred non-alpha amino acids include:

-   8-amino-3,6-dioxaoctanoic acid;-   N-4-aminoethyl-N-1-acetic acid; and-   polyethylene glycol derivatives having the formula    NH₂—(CH₂CH₂O)n-CH₂CO₂H or NH₂—(CH₂CH₂O)n-CH₂CH₂CO₂H where n=2 to    100.

Examples of compounds having the formula M-N—O—P-G which contain linkerswith at least one non-alpha amino acid are listed in Table 1.

TABLE 1 Compounds Containing Linkers With At Least One Non-alpha AminoAcid HPLC Compound method¹ HPLC RT² MS³ IC50⁵ M N O P G* L1 10-40% B5.43 1616.6 5 N,N- Lys 8-amino-3,6- none BBN(7-14) dimethylglycine-Ser-dioxaoctanoic Cys(Acm)-Gly acid L2 10-40% B 5.47 1644.7 3 N,N- Arg8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoicSer-Cys(Acm)-Gly acid L3 10-40% B 5.97 1604.6 >50 N,N- Asp 8-amino-3,6-none BBN(7-14) dimethylglycine- dioxaoctanoic Ser-Cys(Acm)-Gly acid L410-40% B 5.92 1575.5 4 N,N- Ser 8-amino-3,6- none BBN(7-14)dimethylglycine- dioxaoctanoic Ser-Cys(Acm)-Gly acid L5 10-40% B 5.941545.5 9 N,N- Gly 8-amino-3,6- none BBN(7-14) dimethylglycine-dioxaoctanoic Ser-Cys(Acm)-Gly acid L6 10-30% B 7.82 1639 (M + Na) >50N,N- Glu 8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoicSer-Cys(Acm)-Gly acid L7 10-30% B 8.47 1581 (M + Na) 7 N,N- Dala8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoicSer-Cys(Acm)-Gly acid L8 10-30% B 6.72 1639 (M + Na) 4 N,N- 8- Lys noneBBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoicacid L9 10-30% B 7.28  823.3 (M + 2/2) 6 N,N- 8- Arg none BBN(7-14)dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L1010-30% B 7.94 1625.6 (M + Na) >50 N,N- 8- Asp none BBN(7-14)dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L1110-30% B 7.59 1575.6 36 N,N- 8- Ser none BBN(7-14) dimethylglycine-amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L12 10-30% B 7.65 1567.5(M + Na) >50 N,N- 8- Gly none BBN(7-14) dimethylglycine- amino-Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L13 10-30% B 7.86 1617.7 >50N,N- 8- Glu none BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-dioxaoctanoic acid L14 10-30% B 7.9 1581.7 (M + Na) 11 N,N- 8- Dala noneBBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoicacid L15 10-30% B 7.84 1656.8 (M + Na) 11.5 N,N- 8- 8-amino-3,6- noneBBN(7-14) dimethylglycine- amino- dioxaoctanoic Ser-Cys(Acm)-Gly 3,6-acid dioxaoctanoic acid L16 10-30% B 6.65 1597.4 (M + Na) 17 N,N- 8-2,3- none BBN(7-14) dimethylglycine- amino- diaminopropionicSer-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L17 10-30% B 7.6 1488.6 8N,N- none 8-amino-3,6- none BBN(7-14) dimethylglycine- dioxaoctanoicSer-Cys(Acm)-Gly acid L18 10-30% B 7.03 1574.6 7.8 N,N- 2,3-8-amino-3,6- none BBN(7-14) dimethylglycine- diaminopropionicdioxaoctanoic Ser-Cys(Acm)-Gly acid acid L19 10-35% B 5.13 1603.6 >50N,N- Asp 8-amino-3,6- Gly BBN(7-14) dimethylglycine- dioxaoctanoicSer-Cys(Acm)-Gly acid L20 10-35% B 5.19 1603.6 37 N,N- 8- Asp GlyBBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoicacid L21 10-35% B 5.04 1575.7 46 N,N- 8- Ser Gly BBN(7-14)dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L2210-35% B 4.37 1644.7 36 N,N- 8- Arg Gly BBN(7-14) dimethylglycine-amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L23 10-35% B 5.321633.7 >50 N,N- 8- 8-amino-3,6- Gly BBN(7-14) dimethylglycine- amino-dioxaoctanoic Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L24 10-35% B4.18 1574.6 38 N,N- 8- 2,3- Gly BBN(7-14) dimethylglycine- amino-diaminopropionic Ser-Cys(Acm)-Gly 3,6- acid dioxaoctanoic acid L2510-35% B 4.24 1616.6 26 N,N- 8- Lys Gly BBN(7-14) dimethylglycine-amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L26 10-35% B 4.45 1574.630 N,N- 2,3- 8-amino-3,6- Gly BBN(7-14) dimethylglycine-diaminopropionic dioxaoctanoic Ser-Cys(Acm)-Gly acid acid L27 10-35% B4.38 1627.3 >50 N,N- N-4- Asp none BBN(7-14) dimethylglycine-aminoethyl- Ser-Cys(Acm)-Gly N- 1- piperazineacetic acid L28 10-35% B4.1 1600.3 25 N,N- N-4- Ser none BBN(7-14) dimethylglycine- aminoethyl-Ser-Cys(Acm)-Gly N- 1- piperazineacetic acid L29 10-35% B 3.71 1669.4 36N,N- N-4- Arg none BBN(7-14) dimethylglycine- aminoethyl-Ser-Cys(Acm)-Gly N- 1- piperazineacetic acid L30 10-35% B 4.57 1657.2 36N,N- N-4- 8-amino-3,6- none BBN(7-14) dimethylglycine- aminoethyl-dioxaoctanoic Ser-Cys(Acm)-Gly N- acid 1- piperazineacetic acid L3110-35% B 3.69 1598.3 >50 N,N- N-4- 2,3- none BBN(7-14) dimethylglycine-aminoethyl- diaminopropionic Ser-Cys(Acm)-Gly N- acid 1-piperazineacetic acid L32 10-35% B 3.51 1640.3 34 N,N- N-4- Lys noneBBN(7-14) dimethylglycine- aminoethyl- Ser-Cys(Acm)-Gly N- 1-piperazineacetic acid L33 10-35% B 4.29 1584.5 >50 N,N- N-1- Asp noneBBN(7-14) dimethylglycine- piperazineacetic Ser-Cys(Acm)-Gly acid L3410-35% B 4.07 1578.7 (M + Na) 38 N,N- N-1- Ser none BBN(7-14)dimethylglycine- piperazineacetic Ser-Cys(Acm)-Gly acid L35 10-35% B3.65 1625.6 26 N,N- N-1- Arg none BBN(7-14) dimethylglycine-piperazineacetic Ser-Cys(Acm)-Gly acid L36 10-35% B 4.43 1636.6 7 N,N-N-1- 8-amino-3,6- none BBN(7-14) dimethylglycine- piperazineaceticdioxaoctanoic Ser-Cys(Acm)-Gly acid acid L37 10-35% B 3.66 1555.7 23N,N- N-1- 2,3- none BBN(7-14) dimethylglycine- piperazineaceticdiaminopropionic Ser-Cys(Acm)-Gly acid acid L38 10-35% B 3.44 1619.6 7N,N- N-1- Lys none BBN(7-14) dimethylglycine- piperazineaceticSer-Cys(Acm)-Gly acid L42 30-50% B 5.65 1601.6 25 N,N- 4- 8-amino-3,6-none BBN(7-14) dimethylglycine- Hydroxyproline dioxaoctanoicSer-Cys(Acm)-Gly acid L48 30-50% B 4.47 1600.5 40 N,N- 4- 8-amino-3,6-none BBN(7-14) dimethylglycine- aminoproline dioxaoctanoicSer-Cys(Acm)-Gly acid L51 15-35% B 5.14 1673.7 49 N,N- Lys 8-amino-3,6-Gly BBN(7-14) dimethylglycine- dioxaoctanoic Ser-Cys(Acm)-Gly acid L5215-35% B 6.08 1701.6 14 N,N- Arg 8-amino-3,6- Gly BBN(7-14)dimethylglycine- dioxaoctanoic Ser-Cys(Acm)-Gly acid L53 15-35% B 4.161632.6 10 N,N- Ser 8-amino-3,6- Gly BBN(7-14) dimethylglycine-dioxaoctanoic Ser-Cys(Acm)-Gly acid L54 15-35% B 4.88 1661.6 >50 N,N-Asp 8-amino-3,6- Gly BBN(7-14) dimethylglycine- dioxaoctanoicSer-Cys(Acm)-Gly acid L55 15-35% B 4.83 1683.4 (M + Na) 43 N,N- 8- AspGly BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-dioxaoctanoic acid L56 15-35% B 4.65 1655.7 (M + Na) 4 N,N- 8- Ser GlyBBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoicacid L57 15-35% B 4.9 1701.8 50 N,N- 8- Arg Gly BBN(7-14)dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6- dioxaoctanoic acid L5815-35% B 4.22  846.4 (M + H/2) >50 N,N- 8- 8-amino-3,6- Gly BBN(7-14)dimethylglycine- amino- dioxaoctanoic Ser-Cys(Acm)-Gly 3,6- aciddioxaoctanoic acid L59 15-35% B 4.03 1635.5 42 N,N- 8- 2,3- GlyBBN(7-14) dimethylglycine- amino- diaminopropionic Ser-Cys(Acm)-Gly 3,6-acid dioxaoctanoic acid L60 15-35% B 4.11 1696.6 (M + Na) 20 N,N- 8- LysGly BBN(7-14) dimethylglycine- amino- Ser-Cys(Acm)-Gly 3,6-dioxaoctanoic acid L61 15-35% B 4.32 1631.4 43 N,N- 2,3- 8-amino-3,6-Gly BBN(7-14) dimethylglycine- diaminopropionic dioxaoctanoicSer-Cys(Acm)-Gly acid acid L78 20-40% B 6.13 1691.4 (M + Na) 35DO3A-monoamide 8- Diaminopropionic none BBN(7-14) amino- acid 3,6-dioxaoctanoic acid L79 20-40% B 7.72 1716.8 (M + Na) 42 DO3A-monoamide8- Biphenylalanine none BBN(7-14) amino- 3,6- dioxaoctanoic acid L8020-40% B 7.78 1695.9 >50 DO3A-monoamide 8- Diphenylalanine noneBBN(7-14) amino- 3,6- dioxaoctanoic acid L81 20-40% B 7.57 1513.6 37.5DO3A-monoamide 8- 4- none BBN(7-14) amino- Benzoylphenyl 3,6- alaninedioxaoctanoic acid L92 15-30% B 5.63 1571.6 5 DO3A-monoamide 5-8-amino-3,6- none BBN(7-14) aminopentanoic dioxaoctanoic acid acid L9420-36% B 4.19 1640.8 (M + Na) 6.2 DO3A-monoamide 8- D- none BBN(7-14)amino- Phenylalanine 3,6- dioxaoctanoic acid L110 15-45% B 5.06 1612.736 DO3A-monoamide 8- 8-amino-3,6- none BBN(7-14) aminooctanoicdioxaoctanoic acid acid *BBN(7-14) is [SEQ ID NO: 1] ¹HPLC method refersto the 10 minute time for the HPLC gradient. ²HPLC RT refers to theretention time of the compound in the HPLC. ³MS refers to mass spectrawhere molecular weight is calculated from mass/unit charge (m/e). ⁴IC₅₀refers to the concentration of compound to inhibit 50% binding ofiodinated bombesin to a GRP receptor on cells.

2B. Linkers Containing at Least One Substituted Bile Acid

In another embodiment of the present invention, the linker N—O—Pcontains at least one substituted bile acid. Thus, in this embodiment ofthe linker N—O—P,

-   -   N is 0 (where 0 means it is absent), an alpha amino acid, a        substituted bile acid or other linking group;    -   O is an alpha amino acid or a substituted bile acid; and    -   P is 0, an alpha amino acid, a substituted bile acid or other        linking group,    -   wherein at least one of N, O or P is a substituted acid.

Bile acids are found in bile (a secretion of the liver) and are steroidshaving a hydroxyl group and a five carbon atom side chain terminating ina carboxyl group. In substituted bile acids, at least one atom such as ahydrogen atom of the bile acid is substituted with another atom,molecule or chemical group. For example, substituted bile acids includethose having a 3-amino, 24-carboxyl function optionally substituted atpositions 7 and 12 with hydrogen, hydroxyl or keto functionality.

Other useful substituted bile acids in the present invention includesubstituted cholic acids and derivatives thereof. Specific substitutedcholic acid derivatives include:

-   (3β,5β)-3-aminocholan-24-oic acid;-   (3β,5β,12α)-3-amino-12-hydroxycholan-24-oic acid;-   (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid;-   Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholic    acid);-   (3β,5β,7α)-3-amino-7-hydroxy-12-oxocholan-24-oic acid; and-   (3β,5β,7α)-3-amino-7-hydroxycholan-24-oic acid.

Examples of compounds having the formula M-N—O—P-G which contain linkerswith at least one substituted bile acid are listed in Table 2.

TABLE 2 Compounds Containing Linkers With At Least One Substituted BileAcid HPLC HPLC Compound method¹ RT² MS³ IC50⁵ M N O P G* L62 20-80% B3.79 1741.2 >50 DO3A- Gly (3β,5β)-3- none BBN(7-14) monoamideaminocholan- 24-oic acid L63 20-80% B 3.47 1757.0 23 DO3A- Gly(3β,5β,12α)-3- none BBN(7-14) monoamide amino-12- hydroxycholan- 24-oicacid L64 20-50% B 5.31 1773.7 8.5 DO3A- Gly (3β,5β,7α,12α)- noneBBN(7-14) monoamide 3-amino- 7,12- dihydroxycholan- 24-oic acid L6520-80% B 3.57 2246.2 >50 DO3A- Gly Lys-(3,6,9- Arg BBN(7-14) monoamidetrioxaundecane- 1,11- dicarbonyl- 3,7- dideoxy-3- aminocholic acid) L6620-80% 3.79 2245.8 >50 (3β,5β,7α,12α)-3- Lys(DO3A- Arg BBN(7-14)amino-7,12- monoamide- dihydroxycholan-24-oic Gly) acid-3,6,9-trioxaundecane-1,11- dicarbonyl L67 20-80% 3.25 1756.9 4.5 DO3A- Gly(3β,5β,7α,12α)- none BBN(7-14) monoamide 3-amino-12- oxacholan-24- oicacid L69 20-80% 3.25 1861.27 not DO3A- 1- (3β,5β,7α,12α)- none BBN(7-14)tested monoamide amino- 3-amino- 3,6- 7,12- dioxaoctanoicdihydroxycholan- acid 24-oic acid *BBN(7-14) is [SEQ ID NO: 1] ¹HPLCmethod refers to the 10 minute time for the HPLC gradient. ²HPLC RTrefers to the retention time of the compound in the HPLC. ³MS refers tomass spectra where molecular weight is calculated from mass/unit charge(m/e). ⁴IC₅₀ refers to the concentration of compound to inhibit 50%binding of iodinated bombesin to a GRP receptor on cells.

2C. Linkers Containing at Least One Non-Alpha Amino Acid with a CyclicGroup

In yet another embodiment of the present invention, the linker N—O—Pcontains at least one non-alpha amino acid with a cyclic group. Thus, inthis embodiment of the linker N—O—P,

-   -   N is 0 (where 0 means it is absent), an alpha amino acid, a        non-alpha amino acid with a cyclic group or other linking group;    -   O is an alpha amino acid or a non-alpha amino acid with a cyclic        group; and    -   P is 0, an alpha amino acid, a non-alpha amino acid with a        cyclic group, or other linking group,    -   wherein at least one of N, O or P is a non-alpha amino acid with        a cyclic group.

Non-alpha amino acids with a cyclic group include substituted phenyl,biphenyl, cyclohexyl or other amine and carboxyl containing cyclicaliphatic or heterocyclic moieties. Examples of such include:

-   4-aminobenzoic acid-   4-aminomethyl benzoic acid-   trans-4-aminomethylcyclohexane carboxylic acid-   4-(2-aminoethoxy)benzoic acid-   isonipecotic acid-   2-aminomethylbenzoic acid-   4-amino-3-nitrobenzoic acid-   4-(3-carboxymethyl-2-keto-1-benzimidazolyl-piperidine-   6-(piperazin-1-yl)-4-(3H)-quinazolinone-3-acetic acid-   (2S,5S)-5-amino-1,2,4,5,6,7-hexahydro-azepino[3,21-hi]indole-4-one-2-carboxylic    acid-   (4S,7R)-4-amino-6-aza-5-oxo-9-thiabicyclo[4.3.0]nonane-7-carboxylic    acid-   3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one-   N1-piperazineacetic acid-   N-4-aminoethyl-N-1-piperazineacetic acid-   (3S)-3-amino-1-carboxymethylcaprolactam-   (2S,6S,9)-6-amino-2-carboxymethyl-3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione

Examples of compounds having the formula M-N—O—P-G which contain linkerswith at least one alpha amino acid with a cyclic group are listed inTable 3.

TABLE 3 Compounds Containing Linkers Related To Amino-(Phenyl, Biphenyl,Cycloalkyl Or Heterocyclic) Carboxylates HPLC Compound method¹ HPLC RT²MS³ IC50⁵ M N O P G* L70 10-40% B 6.15 1502.6 5 DO3A- Gly 4-aminobenzoicnone BBN(7-14) monoamide acid L71 1482.2 (M + Na) 7 DO3A- none4-aminomethyl none BBN(7-14) monoamide benzoic acid L72 1504.0 (M + K) 8DO3A- none trans-4- none BBN(7-14) monoamide aminomethylcyclohexylcarboxylic acid L73  5-35% 7.01 1489.8 5 DO3A- none 4-(2- none BBN(7-14)monoamide aminoethoxy)benzoic acid L74  5-35% 6.49 1494.8 7 DO3A- Glyisonipecotic none BBN(7-14) monoamide acid L75  5-35% 6.96 1458.0 23DO3A- none 2- none BBN(7-14) monoamide aminomethylbenzoic acid L76 5-35% 7.20] 1502.7 4 DO3A- none 4-aminomethyl- none BBN(7-14) monoamide3-nitrobenzoic acid L77 20-40% B 6.17 1691.8 (M + Na) 17.5 DO3A- 8- 1-none BBN(7-14) monoamide amino- Naphthylalanine 3,6- dioxaoctanoic acidL82 20-40% B 6.18 1584.6 8 DO3A- none 4-(3- none BBN(7-14) monoamidecarboxymethyl- 2-keto-1- benzimidazolyl- piperidine L83 20-40% B 5.661597.5 >50 DO3A- none 6-(piperazin-1- none BBN(7-14) monoamideyl)-4-(3H)- quinazolinone- 3-acetic acid L84 20-40% B 6.31 1555.5 >50DO3A- none (2S,5S)-5- none BBN(7-14) monoamide amino- 1,2,4,5,6,7-hexahydro- azepino[3,21- hi]indole- 4-one-2- carboxylic acid L85 20-40%B 5.92 1525.5 >50 DO3A- none (4S,7R)-4- none BBN(7-14) monoamideamino-6-aza-5- oxo-9- thiabicyclo[4.3. 0]nonane-7- carboxylic acid L8720-40% B 5.47 1593.8 (M + Na) >50 DO3A- none 3- none BBN(7-14) monoamidecarboxymethyl- 1-phenyl-1,3,8- triazaspiro[4.5] decan-4-one L88 20-40% B3.84 1452.7 >50 DO3A- none N1- none BBN(7-14) monoamide piperazineaceticacid L89 20-40% B 5.68 1518.5 (M + Na) 23 DO3A- none N-4- none BBN(7-14)monoamide aminoethyl-N- 1-piperazine- acetic acid L90 20-40% B 7.951495.4 50 DO3A- none (3S)-3-amino- none BBN(7-14) monoamide1-carboxymethylcaprolactam L91 20-40% B 3.97 1535.7 >50 DO3A- none(2S,6S,9)-6- none BBN(7-14) monoamide amino-2- carboxymethyl- 3,8-diazabicyclo- [4,3,0]-nonane- 1,4-dione L93 15-30% B 7.57 1564.7 5.8DO3A- 5- trans-4- none BBN(7-14) monoamide aminopentanoicaminomethylcyclohexane- acid 1- carboxylic acid L95 15-35% B 5.41 1604.614 DO3A- trans-4- D- none BBN(7-14) monoamide amino Phenylalanine methylcyclohexane- 1- carboxylic acid L96 20-36% B 4.75 1612.7 35 DO3A- 4-8-amino-3,6- none BBN(7-14) monoamide amino dioxaoctanoic methyl acidbenzoic acid L97 15-35% B 5.86 1598.8 4.5 DO3A- 4- trans-4- noneBBN(7-14) monoamide benzoyl- aminomethylcyclohexane- (L)- 1- phenylcarboxylic acid alanine L98 15-35% B 4.26 1622.7 16 DO3A- trans-4- Argnone BBN(7-14) monoamide amino methyl cyclohexane- 1- carboxylic acidL99 15-35% B 4.1 1594.7 22 DO3A- trans-4- Lys none BBN(7-14) monoamideamino methyl cyclohexane- 1- carboxylic acid L100 15-35% B 4.18 1613.610 DO3A- trans-4- Diphenylalanine none BBN(7-14) monoamide amino methylcyclohexane- 1- carboxylic acid L101 15-35% B 5.25 1536.7 25 DO3A-trans-4- 1- none BBN(7-14) monoamide amino Naphthylalanine methylcyclohexane- 1- carboxylic acid L102 15-35% B 5.28 1610.8 9.5 DO3A-trans-4- 8-amino-3,6- none BBN(7-14) monoamide amino dioxaoctanoicmethyl acid cyclohexane- 1- carboxylic acid L103 15-35% B 4.75 1552.7 24DO3A- trans-4- Ser none BBN(7-14) monoamide amino methyl cyclohexane- 1-carboxylic acid L104 15-35% B 3.91 1551.7 32 DO3A- trans-4- 2,3- noneBBN(7-14) monoamide amino diaminopropionic methyl acid cyclohexane- 1-carboxylic acid L105 20-45% B 7.68 1689.7 3.5 DO3A- trans-4-Biphenylalanine none BBN(7-14) monoamide amino methyl cyclohexane- 1-carboxylic acid L106 20-45% B 6.97 1662.7 3.8 DO3A- trans-4- (2S,5S)-5-none BBN(7-14) monoamide amino amino- methyl 1,2,4,5,6,7- cyclohexane-hexahydro- 1- azepino[3,21- carboxylic hi]indole- acid 4-one-2-carboxylic acid L107 15-35% B 5.79 1604.7 5 DO3A- trans-4- trans-4- noneBBN(7-14) monoamide amino aminomethylcyclohexane- methyl 1- cyclohexane-carboxylic acid 1- carboxylic acid L108 15-45% B 6.38 1618.7 10 DO3A- 8-Phenylalanine none BBN(7-14) monoamide amino- 3,6- dioxaoctanoic acidL109 15-45% B 6.85 1612.7 6 DO3A- trans-4- Phenylalanine none BBN(7-14)monoamide amino methyl cyclohexane- 1- carboxylic acid L111 20-45% B3.75 1628.6 8 DO3A- 8- trans-4- none BBN(7-14) monoamide aminooctanoicaminomethylcyclohexane- acid 1- carboxylic acid L112 20-47% B 3.6 1536.54.5 DO3A- none 4′- none BBN(7-14) in 9 min monoamide aminomethyl-biphenyl-1- carboxylic acid L113 20-47% B 3.88 1558.6 (M + Na) 5 DO3A-none 3′- none BBN(7-14) in 9 min monoamide aminomethyl- biphenyl-3-carboxylic acid L114 10-40% B 5.47 1582.8 4.5 CMDOTA Gly 4-aminobenzoicnone BBN(7-14) acid L124  5-35% B 7.04 1489.9 8.0 DO3A- none 4- noneBBN(7-14) monoamide aminomethylphenoxyacetic acid L143  5-35% B 6.851516.8 NT** DO3A- Gly 4- none BBN(7-14) monoamide aminophenylacetic acidL144  5-35% B 6.85 1462.7 NT HPDO3A none 4-phenoxy none BBN(7-14) L14520-80% B 1.58 1459.8 5 DO3A- none 3- none BBN(7-14) monoamideaminomethylbenzoic acid L146 20-80% B 1.53 1473.7 9 DO3A- none 4- noneBBN(7-14) monoamide aminomethylphenylacetic acid L147 20-80% B 1.681489.7 NT DO3A- none 4-aminomethyl- none BBN(7-14) monoamide 3-methoxybenzoic acid *BBN(7-14) is [SEQ ID NO: 1] **NT is defined as “nottested.” ¹HPLC method refers to the 10 minute time for the HPLCgradient. ²HPLC RT refers to the retention time of the compound in theHPLC. ³MS refers to mass spectra where molecular weight is calculatedfrom mass/unit charge (m/e). ⁴IC₅₀ refers to the concentration ofcompound to inhibit 50% binding of iodinated bombesin to a GRP receptoron cells.

2D. Other Linking Groups

Other linking groups which may be used within the linker N—O—P include achemical group that serves to couple the GRP receptor targeting peptideto the metal chelator while not adversely affecting either the targetingfunction of the GRP receptor targeting peptide or the metal complexingfunction of the metal chelator. Suitable other linking groups includepeptides (i.e., amino acids linked together) alone, a non-peptide group(e.g., hydrocarbon chain) or a combination of an amino acid sequence anda non-peptide spacer.

In one embodiment, other linking groups for use within the linker N—O—Pinclude L-glutamine and hydrocarbon chain, or a combination thereof.

In another embodiment, other linking groups for use within the linkerN—O—P include a pure peptide linking group consisting of a series ofamino acids (e.g., diglycine, triglycine, gly-gly-glu, gly-ser-gly,etc.), in which the total number of atoms between the N-terminal residueof the GRP receptor targeting peptide and the metal chelator in thepolymeric chain is <12 atoms.

In yet a further embodiment, other linking groups for use within thelinker N—O—P can also include a hydrocarbon chain [i.e.,R₁—(CH₂)_(n)—R₂] wherein n is 0-10, preferably n=3 to 9, R₁ is a group(e.g., H₂N—, HS—, —COOH) that can be used as a site for covalentlylinking the ligand backbone or the preformed metal chelator or metalcomplexing backbone; and R₂ is a group that is used for covalentcoupling to the N-terminal NH₂-group of the GRP receptor targetingpeptide (e.g., R₂ is an activated COOH group). Several chemical methodsfor conjugating ligands (i.e., chelators) or preferred metal chelates tobiomolecules have been well described in the literature [Wilbur, 1992;Parker, 1990; Hermanson, 1996; Frizberg et al., 1995]. One or more ofthese methods could be used to link either the uncomplexed ligand(chelator) or the radiometal chelate to the linker or to link the linkerto the GRP receptor targeting peptides. These methods include theformation of acid anhydrides, aldehydes, arylisothiocyanates, activatedesters, or N-hydroxysuccinimides [Wilbur, 1992; Parker, 1990; Hermanson,1996; Frizberg et al., 1995].

In a preferred embodiment, other linking groups for use within thelinker N—O—P may be formed from linker precursors having electrophilesor nucleophiles as set forth below:

-   -   LP1: a linker precursor having on at least two locations of the        linker the same electrophile E1 or the same nucleophile Nu1;    -   LP2: a linker precursor having an electrophile E1 and on another        location of the linker a different electrophile E2;    -   LP3: a linker precursor having a nucleophile Nu1 and on another        location of the linker a different nucleophile Nu2; or    -   LP4: a linker precursor having one end functionalized with an        electrophile E1 and the other with a nucleophile Nu1.

The preferred nucleophiles Nu1/Nu2 include —OH, —NH, —NR, —SH, —HN—NH₂,—RN—NH₂, and —RN—NHR′, in which R′ and R are independently selected fromthe definitions for R given above, but for R′ is not H.

The preferred electrophiles E1/E2 include —COOH, —CH═O (aldehyde),—CR═OR′ (ketone), —RN—C═S, —RN—C═O, —S—S-2-pyridyl, —SO₂—Y, —CH₂C(═O)Y,and

wherein Y can be selected from the following groups:

3. GRP Receptor Targeting Peptide

The GRP receptor targeting peptide (i.e., G in the formula M-N—O—P-G) isany peptide, equivalent, derivative or analogue thereof which has abinding affinity for the GRP receptor family.

The GRP receptor targeting peptide may take the form of an agonist or anantagonist. A GRP receptor targeting peptide agonist is known to“activate” the cell following binding with high affinity and may beinternalized by the cell. Conversely, GRP receptor targeting peptideantagonists are known to bind only to the GRP receptor on the cellwithout being internalized by the cell and without “activating” thecell. In a preferred embodiment, the GRP receptor targeting peptide isan agonist.

In a more preferred embodiment of the present invention, the GRP agonistis a bombesin (BBN) analogue and/or a derivative thereof. The BBNderivative or analog thereof preferably contains either the same primarystructure of the BBN binding region (i.e., BBN(7-14) [SEQ ID NO:1]) orsimilar primary structures, with specific amino acid substitutions thatwill specifically bind to GRP receptors with better or similar bindingaffinities as BBN alone (i.e., Kd<25 nM). Suitable compounds includepeptides, peptidomimetics and analogues and derivatives thereof. Thepresence of L-methionine (Met) at position BBN-14 will generally conferagonistic properties while the absence of this residue at BBN-14generally confers antagonistic properties [Hoffken, 1994].

It is well documented in the art that there are a few and selectivenumber of specific amino acid substitutions in the BBN (8-14) bindingregion (e.g., D-Ala¹¹ for L-Gly¹¹ or D-Trp⁸ for L-Trp⁸), which can bemade without decreasing binding affinity [Leban et al., 1994; Qin etal., 1994; Jensen et al., 1993]. In addition, attachment of some aminoacid chains or other groups to the N-terminal amine group at positionBBN-8 (i.e., the Trp⁸ residue) can dramatically decrease the bindingaffinity of BBN analogues to GRP receptors [Davis et al., 1992; Hoffken,1994; Moody et al., 1996; Coy, et al., 1988; Cai et al., 1994]. In a fewcases, it is possible to append additional amino acids or chemicalmoieties without decreasing binding affinity.

Analogues of BBN receptor targeting peptides include molecules thattarget the GRP receptors with avidity that is greater than or equal toBBN, as well as muteins, retropeptides and retro-inverso-peptides of GRPor BBN. One of ordinary skill will appreciate that these analogues mayalso contain modifications which include substitutions, and/or deletionsand/or additions of one or several amino acids, insofar that thesemodifications do not negatively alter the biological activity of thepeptides described therein. These substitutions may be carried out byreplacing one or more amino acids by their synonymous amino acids.Synonymous amino acids within a group are defined as amino acids thathave sufficient physicochemical properties to allow substitution betweenmembers of a group in order to preserve the biological function of themolecule. Synonymous amino acids as used herein include syntheticderivatives of these amino acids and may include those listed in thefollowing Table. In the chart and throughout this application aminoacids are abbreviated interchangeably either by their three letter orsingle letter abbreviations, which are well known to the skilledartisan. Thus, for example, T or Thr stands for threonine, K or Lysstands for lysine, P or Pro stands for proline and R or Arg stands forarginine.

Amino Acids Synonymous Groups Arg His, Lys, Glu, Gln Pro Ala, Thr, Gly,N-methyl Ala, pipecolic acid, azetidine carboxylic acid Thr 3-hydroxyproline, 4-hydroxy proline, Ser, Ala, Gly, His, Gln Lys Lys, ornithine,Arg, diaminopropionic acid, HArg, His

Deletions or insertions of amino acids may also be introduced into thedefined sequences provided they do not alter the biological functions ofsaid sequences. Preferentially such insertions or deletions should belimited to 1, 2, 3, 4 or 5 amino acids and should not remove orphysically disturb or displace amino acids which are critical to thefunctional conformation. Muteins of the GRP receptor targeting peptidesdescribed herein may have a sequence homologous to the sequencedisclosed in the present specification in which amino acidsubstitutions, deletions, or insertions are present at one or more aminoacid positions. Muteins may have a biological activity that is at least40%, preferably at least 50%, more preferably 60-70%, most preferably80-90% of the peptides described herein. However, they may also have abiological activity greater than the peptides specifically exemplified,and thus do not necessarily have to be identical to the biologicalfunction of the exemplified peptides. Analogues of GRP receptortargeting peptides also include peptidomimetics or pseudopeptidesincorporating changes to the amide bonds of the peptide backbone,including thioamides, methylene amines, and E-olefins. Also peptidesbased on the structure of GRP, BBN or their peptide analogues with aminoacids replaced by N-substituted hydrazine carbonyl compounds (also knownas aza amino acids) are included in the term analogues as used herein.

The GRP receptor targeting peptide can be prepared by various methodsdepending upon the selected chelator. The peptide can generally be mostconveniently prepared by techniques generally established and known inthe art of peptide synthesis, such as the solid-phase peptide synthesis(SPPS) approach. Solid-phase peptide synthesis (SPPS) involves thestepwise addition of amino acid residues to a growing peptide chain thatis linked to an insoluble support or matrix, such as polystyrene. TheC-terminal residue of the peptide is first anchored to a commerciallyavailable support with its amino group protected with an N-protectingagent such as a t-butyloxycarbonyl group (Boc) or afluorenylmethoxycarbonyl (Fmoc) group. The amino protecting group isremoved with suitable deprotecting agents such as TFA in the case of Bocor piperidine for Fmoc and the next amino acid residue (in N-protectedform) is added with a coupling agent such asN,N′-dicyclohexylcarbodiimide (DCC), or N,N′-diisopropylcarbodiimide(DIC) or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU). Upon formation of a peptide bond, thereagents are washed from the support. After addition of the finalresidue, the peptide is cleaved from the support with a suitable reagentsuch as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

The linker may then be coupled to form a conjugate by reacting the freeamino group of the Trp⁸ residue of the GRP receptor targeting peptidewith an appropriate functional group of the linker. The entire constructof chelator, linker and targeting moiety discussed above may also beassembled on resin and then cleaved by agency of suitable reagents suchas trifluoroacetic acid or HF, as well.

4. Labeling and Administration of Compounds

Incorporation of the metal within the conjugate can be achieved byvarious methods commonly known in the art of coordination chemistry.When the metal is ^(99m)Tc, a preferred radionuclide for diagnosticimaging, the following general procedure can be used to form atechnetium complex. A peptide-chelator conjugate solution is formed byinitially dissolving the conjugate in water, dilute acid, or in anaqueous solution of an alcohol such as ethanol. The solution is thenoptionally degassed to remove dissolved oxygen. When an —SH group ispresent in the peptide, a thiol protecting group such as Acm(acetamidomethyl), trityl or other thiol protecting group may optionallybe used to protect the thiol from oxidation. The thiol protectinggroup(s) are removed with a suitable reagent, for example with sodiumhydroxide, and are then neutralized with an organic acid such as aceticacid (pH 6.0-6.5). Alternatively, the thiol protecting group can beremoved in situ during technetium chelation. In the labeling step,sodium pertechnetate obtained from a molybdenum generator is added to asolution of the conjugate with a sufficient amount of a reducing agent,such as stannous chloride, to reduce technetium and is either allowed tostand at room temperature or is heated. The labeled conjugate can beseparated from the contaminants ^(99m)TcO₄ ⁻ and colloidal ^(99m)TcO₂chromatographically, for example with a C-18 Sep Pak cartridge[Millipore Corporation, Waters Chromatography Division, 34 Maple Street,Milford, Mass. 01757] or by HPLC using methods known to those skilled inthe art.

In an alternative method, the labeling can be accomplished by atranschelation reaction. In this method, the technetium source is asolution of technetium that is reduced and complexed with labile ligandsprior to reaction with the selected chelator, thus facilitating ligandexchange with the selected chelator. Examples of suitable ligands fortranschelation includes tartrate, citrate, gluconate, andheptagluconate. It will be appreciated that the conjugate can be labeledusing the techniques described above, or alternatively, the chelatoritself may be labeled and subsequently coupled to the peptide to formthe conjugate; a process referred to as the “prelabeled chelate” method.Re and Tc are both in row VIIB of the Periodic Table and they arechemical congeners. Thus, for the most part, the complexation chemistryof these two metals with ligand frameworks that exhibit high in vitroand in vivo stabilities are the same [Eckelman, 1995] and similarchelators and procedures can be used to label with Re. Many ^(99m)Tc or^(186/188)Re complexes, which are employed to form stable radiometalcomplexes with peptides and proteins, chelate these metals in their +5oxidation state [Lister-James et al., 1997]. This oxidation state makesit possible to selectively place ^(99m)Tc- or ^(186/188)Re into ligandframeworks already conjugated to the biomolecule, constructed from avariety of ^(99m)Tc(V) and/or ^(186/188)Re(V) weak chelates (e.g.,^(99m)Tc-glucoheptonate, citrate, gluconate, etc.) [Eckelman, 1995;Lister-James et al., 1997; Pollak et al., 1996].

5. Diagnostic and Therapeutic Uses

When labeled with diagnostically and/or therapeutically useful metals,compounds of the present invention can be used to treat and/or detectcancers, including tumors, by procedures established in the art ofradiodiagnostics and radiotherapeutics. [Bushbaum, 1995; Fischman etal., 1993; Schubiger et al., 1996; Lowbertz et al., 1994; Krenning etal., 1994].

The compounds of the invention, which, as explained in more detail inthe Examples, show higher uptake in tumors in vivo than compoundswithout the novel linkers disclosed herein, exhibit an improved abilityto target GRP receptor-expressing tumors and thus to image or deliverradiotherapy to these tissues. Indeed, as shown in the Examples,radiotherapy is more effective (and survival time increased) usingcompounds of the invention.

The diagnostic application of these compounds can be as a first linediagnostic screen for the presence of neoplastic cells usingscintigraphic imaging, as an agent for targeting neoplastic tissue usinghand-held radiation detection instrumentation in the field ofradioimmuno guided surgery (RIGS), as a means to obtain dosimetry dataprior to administration of the matched pair radiotherapeutic compound,and as a means to assess GRP receptor population as a function oftreatment over time.

The therapeutic application of these compounds can be defined either asan agent that will be used as a first line therapy in the treatment ofcancer, as combination therapy where these radiolabeled agents could beutilized in conjunction with adjuvant chemotherapy, and as the matchedpair therapeutic agent. The matched pair concept refers to a singleunmetallated compound which can serve as both a diagnostic and atherapeutic agent depending on the radiometal that has been selected forbinding to the appropriate chelate. If the chelator cannot accommodatethe desired metals appropriate substitutions can be made to accommodastethe different metal whilst maintaining the pharmacology such that thebehaviour of the diagnostic compound in vivo can be used to predict thebehaviour of the radiotherapeutic compound.

A conjugate labeled with a radionuclide metal, such as ^(99m)Tc, can beadministered to a mammal, including human patients or subjects, byintravenous, subcutaneous or intraperitoneal injection in apharmaceutically acceptable carrier and/or solution such as saltsolutions like isotonic saline. Radiolabeled scintigraphic imagingagents provided by the present invention are provided having a suitableamount of radioactivity. In forming ^(99m)Tc radioactive complexes, itis generally preferred to form radioactive complexes in solutionscontaining radioactivity at concentrations of from about 0.01 millicurie(mCi) to 100 mCi per mL. Generally, the unit dose to be administered hasa radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to30 mCi. The solution to be injected at unit dosage is from about 0.01 mLto about 10 mL. The amount of labeled conjugate appropriate foradministration is dependent upon the distribution profile of the chosenconjugate in the sense that a rapidly cleared conjugate may need to beadministered in higher doses than one that clears less rapidly. In vivodistribution and localization can be tracked by standard scintigraphictechniques at an appropriate time subsequent to administration;typically between thirty minutes and 180 minutes depending upon the rateof accumulation at the target site with respect to the rate of clearanceat non-target tissue. For example, after injection of the diagnosticradionuclide-labeled compounds of the invention into the patient, agamma camera calibrated for the gamma ray energy of the nuclideincorporated in the imaging agent can be used to image areas of uptakeof the agent and quantify the amount of radioactivity present in thesite. Imaging of the site in vivo can take place in a few minutes.However, imaging can take place, if desired, hours or even longer, afterthe radiolabeled peptide is injected into a patient. In most instances,a sufficient amount of the administered dose will accumulate in the areato be imaged within about 0.1 hour to permit the taking of scintiphotos.

The compounds of the present invention can be administered to a patientalone or as part of a composition that contains other components such asexcipients, diluents, radical scavengers, stabilizers, and carriers, allof which are well-known in the art. The compounds can be administered topatients either intravenously or intraperitoneally.

There are numerous advantages associated with the present invention. Thecompounds made in accordance with the present invention form stable,well-defined ^(99m)Tc or ^(186/188)Re labeled compounds. Similarcompounds of the invention can also be made by using appropriatechelator frameworks for the respective radiometals, to form stable,well-defined products labeled with ¹⁵³Sm, ⁹⁰Y, ¹⁶⁶Ho, ¹⁰⁵Rh, ¹⁹⁹Au, ¹⁴⁹Pm, ¹⁷⁷Lu, ¹¹¹In or other radiometal. The radiolabeled GRP receptortargeting peptides selectively bind to neoplastic cells expressing GRPreceptors, and if an agonist is used, become internalized, and areretained in the tumor cells for extended time periods. The radioactivematerial that does not reach (i.e., does not bind) the cancer cells ispreferentially excreted efficiently into the urine with minimalretention of the radiometal in the kidneys.

6. Radiotherapy

Radioisotope therapy involves the administration of a radiolabeledcompound in sufficient quantity to damage or destroy the targetedtissue. After administration of the compound (by e.g., intravenous,subcutaneous, or intraperitonal injection), the radiolabeledpharmaceutical localizes preferentially at the disease site (in thisinstance, tumor tissue that expresses the GRP receptor). Once localized,the radiolabeled compound then damages or destroys the diseased tissuewith the energy that is released during the radioactive decay of theisotope that is administered.

The design of a successful radiotherapeutic involves several criticalfactors:

1. selection of an appropriate targeting group to deliver theradioactivity to the disease site;

2. selection of an appropriate radionuclide that releases sufficientenergy to damage that disease site, without substantially damagingadjacent normal tissues; and

3. selection of an appropriate combination of the targeting group andthe radionuclide without adversely affecting the ability of thisconjugate to localize at the disease site. For radiometals, this ofteninvolves a chelating group that coordinates tightly to the radionuclide,combined with a linker that couples said chelate to the targeting group,and that affects the overall biodistribution of the compound to maximizeuptake in target tissues and minimize uptake in normal, non-targetorgans.

The present invention provides radiotherapeutic agents that satisfy allthree of the above criteria, through proper selection of targetinggroup, radionuclide, metal chelate and linker.

Radiotherapeutic agents may contain a chelated 3+ metal ion from theclass of elements known as the lanthanides (elements of atomic number57-71) and their analogs (i.e. M³⁺ metals such as yttrium and indium).Typical radioactive metals in this class include the isotopes90-Yttrium, 111-Indium, 149-Promethium, 153-Samarium, 166-Dysprosium,166-Holmium, 175-Ytterbium, and 177-Lutetium. All of these metals (andothers in the lanthanide series) have very similar chemistries, in thatthey remain in the +3 oxidation state, and prefer to chelate to ligandsthat bear hard (oxygen/nitrogen) donor atoms, as typified by derivativesof the well known chelate DTPA (diethylenetriaminepentaacetic acid) andpolyaza-polycarboxylate macrocycles such as DOTA(1,4,7,10-tetrazacyclododecane-N,N′,N″,N′″-tetraacetic acid and itsclose analogs. The structures of these chelating ligands, in their fullydeprotonated form are shown below.

These chelating ligands encapsulate the radiometal by binding to it viamultiple nitrogen and oxygen atoms, thus preventing the release of free(unbound) radiometal into the body. This is important, as in vivodissociation of 3⁺ radiometals from their chelate can result in uptakeof the radiometal in the liver, bone and spleen [Brechbiel M W, Gansow OA, “Backbone-substituted DTPA ligands for ⁹⁰Y radioimmunotherapy”,Bioconj. Chem. 1991; 2: 187-194; L1, WP, Ma D S, Higginbotham C, HoffmanT, Ketring A R, Cutler C S, Jurisson, S S, “evelopment of an in vitromodel for assessing the in vivo stability of lanthanide chelates.” Nucl.Med. Biol. 2001; 28(2): 145-154; Kasokat T, Urich K. Arzneim.-Forsch,“Quantification of dechelation of gadopentetate dimeglumine in rats”.1992; 42(6): 869-76]. Unless one is specifically targeting these organs,such non-specific uptake is highly undesirable, as it leads tonon-specific irradiation of non-target tissues, which can lead to suchproblems as hematopoietic suppression due to irradiation of bone marrow.

For radiotherapy applications any of the chelators for therapeuticradionuclides disclosed herein may be used. However, forms of the DOTAchelate [Tweedle M F, Gaughan G T, Hagan J T,“1-Substituted-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecaneand analogs.” U.S. Pat. No. 4,885,363, Dec. 5, 1989] are particularlypreferred, as the DOTA chelate is expected to de-chelate less in thebody than DTPA or other linear chelates.

General methods for coupling DOTA-type macrocycles to targeting groupsthrough a linker (e.g. by activation of one of the carboxylates of theDOTA to form an active ester, which is then reacted with an amino groupon the linker to form a stable amide bond), are known to those skilledin the art. (See e.g. Tweedle et al. U.S. Pat. No. 4,885,363). Couplingcan also be performed on DOTA-type macrocycles that are modified on thebackbone of the polyaza ring.

The selection of a proper nuclide for use in a particularradiotherapeutic application depends on many factors, including:

a. Physical half-life—This should be long enough to allow synthesis andpurification of the radiotherapeutic construct from radiometal andconjugate, and delivery of said construct to the site of injection,without significant radioactive decay prior to injection. Preferably,the radionuclide should have a physical half-life between about 0.5 and8 days.

b. Energy of the emission(s) from the radionuclide—Radionuclides thatare particle emitters (such as alpha emitters, beta emitters and Augerelectron emitters) are particularly useful, as they emit highlyenergetic particles that deposit their energy over short distances,thereby producing highly localized damage. Beta emitting radionuclidesare particularly preferred, as the energy from beta particle emissionsfrom these isotopes is deposited within 5 to about 150 cell diameters.Radiotherapeutic agents prepared from these nuclides are capable ofkilling diseased cells that are relatively close to their site oflocalization, but cannot travel long distances to damage adjacent normaltissue such as bone marrow.

c. Specific activity (i.e. radioactivity per mass of theradionuclide)—Radionuclides that have high specific activity (e.g.generator produced 90-Y, 111-In, 177-Lu) are particularly preferred. Thespecific activity of a radionuclide is determined by its method ofproduction, the particular target that is used to produce it, and theproperties of the isotope in question.

Many of the lanthanides and lanthanoids include radioisotopes that havenuclear properties that make them suitable for use as radiotherapeuticagents, as they emit beta particles. Some of these are listed in thetable below.

Approximate range of b- particle Half-Life Max b-energy Gamma energy(cell Isotope (days) (MeV) (keV) diameters) ¹⁴⁹-Pm 2.21 1.1 286 60¹⁵³-Sm 1.93 0.69 103 30 ¹⁶⁶-Dy 3.40 0.40 82.5 15 ¹⁶⁶-Ho 1.12 1.8 80.6117 ¹⁷⁵-Yb 4.19 0.47 396 17 ¹⁷⁷-Lu 6.71 0.50 208 20 ⁹⁰-Y 2.67 2.28 — 150¹¹¹-In 2.810 Auger electron 173, 247 <5 μm emitter Pm: Promethium, Sm:Samarium, Dy: Dysprosium, Ho: Holmium, Yb: Ytterbium, Lu: Lutetium, Y:Yttrium, In: Indium

Methods for the preparation of radiometals such as beta-emittinglanthanide radioisotopes are known to those skilled in the art, and havebeen described elsewhere [e.g. Cutler C S, Smith C J, Ehrhardt G J.;Tyler T T, Jurisson S S, Deutsch E. “Current and potential therapeuticuses of lanthanide radioisotopes.” Cancer Biother. Radiopharm. 2000;15(6): 531-545]. Many of these isotopes can be produced in high yieldfor relatively low cost, and many (e.g. ⁹⁰-Y, ¹⁴⁹-Pm, ¹⁷⁷-Lu) can beproduced at close to carrier-free specific activities (i.e. the vastmajority of atoms are radioactive). Since non-radioactive atoms cancompete with their radioactive analogs for binding to receptors on thetarget tissue, the use of high specific activity radioisotope isimportant, to allow delivery of as high a dose of radioactivity to thetarget tissue as possible.

Radiotherapeutic derivatives of the invention containing beta-emittingisotopes of rhenium (¹⁸⁶-Re and ¹⁸⁸-Re) are also particularly preferred.

7. Dosages and Additives

Proper dose schedules for the radiopharmaceutical compounds of thepresent invention are known to those skilled in the art. The compoundscan be administered using many methods which include, but are notlimited to, a single or multiple IV or IP injections, using a quantityof radioactivity that is sufficient to permit imaging or, in the case ofradiotherapy, to cause damage or ablation of the targeted GRP-R bearingtissue, but not so much that substantive damage is caused to non-target(normal tissue). The quantity and dose required for scintigraphicimaging is discussed supra. The quantity and dose required forradiotherapy is also different for different constructs, depending onthe energy and half-life of the isotope used, the degree of uptake andclearance of the agent from the body and the mass of the tumor. Ingeneral, doses can range from a single dose of about 30-50 mCi to acumulative dose of up to about 3 Curies.

The radiopharmaceutical compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and may include, for example, para-aminobenzoic acid, ascorbic acid,gentistic acid and the like.

A single, or multi-vial kit that contains all of the components neededto prepare the radiopharmaceuticals of this invention, other than theradionuclide, is an integral part of this invention.

A single-vial kit preferably contains a chelator/linker/targetingpeptide conjugate of the formula M-N—O—P-G, a source of stannous salt(if reduction is required, e.g., when using technetium), or otherpharmaceutically acceptable reducing agent, and is appropriatelybuffered with pharmaceutically acceptable acid or base to adjust the pHto a value of about 3 to about 9. The quantity and type of reducingagent used will depend highly on the nature of the exchange complex tobe formed. The proper conditions are well known to those that areskilled in the art. It is preferred that the kit contents be inlyophilized form. Such a single vial kit may optionally contain labileor exchange ligands such as glucoheptonate, gluconate, mannitol, malate,citric or tartaric acid and can also contain reaction modifiers such asdiethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraaceticacid (EDTA), or α, β, or γ-cyclodextrin that serve to improve theradiochemical purity and stability of the final product. The kit mayalso contain stabilizers, bulking agents such as mannitol, that aredesigned to aid in the freeze-drying process, and other additives knownto those skilled in the art.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial may contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g. the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains thechelator and targeting peptide, as well as buffers appropriate to adjustthe pH to its optimal value. After a reaction time of about 5 to 60minutes, the complexes of the present invention are formed. It isadvantageous that the contents of both vials of this multi-vial kit belyophilized. As above, reaction modifiers, exchange ligands,stabilizers, bulking agents, etc. may be present in either or bothvials.

General Preparation of Compounds

The compounds of the present invention can be prepared by variousmethods depending upon the selected chelator. The peptide portion of thecompound can be most conveniently prepared by techniques generallyestablished and known in the art of peptide synthesis, such as thesolid-phase peptide synthesis (SPPS) approach. Because it is amenable tosolid phase synthesis, employing alternating FMOC protection anddeprotection is the preferred method of making short peptides.Recombinant DNA technology is preferred for producing proteins and longfragments thereof.

Solid-phase peptide synthesis (SPPS) involves the stepwise addition ofamino acid residues to a growing peptide chain that is linked to aninsoluble support or matrix, such as polystyrene. The C-terminal residueof the peptide is first anchored to a commercially available supportwith its amino group protected with an N-protecting agent such as at-butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc)group. The amino protecting group is removed with suitable deprotectingagents such as TFA in the case of Boc or piperidine for Fmoc and thenext amino acid residue (in N-protected form) is added with a couplingagent such as diisopropylcarbodiimide (DIC). Upon formation of a peptidebond, the reagents are washed from the support. After addition of thefinal residue, the peptide is cleaved from the support with a suitablereagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

Alternative Preparation of the Compounds Via Segment Coupling

The compounds of the invention may also be prepared by the process knownin the art as segment coupling or fragment condensation (Barlos, K. andGatos, D.; 2002 “Convergent Peptide Synthesis” in Fmoc Solid PhaseSynthesis—A Practical Approach; Eds. Chan, W. C. and White, P. D.;Oxford University Press, New York; Chap. 9, pp 215-228). In this methodsegments of the peptide usually in side-chain protected form, areprepared separately by either solution phase synthesis or solid phasesynthesis or a combination of the two methods. The choice of segments iscrucial and is made using a division strategy that can provide amanageable number of segments whose C-terminal residues and N-terminalresidues are projected to provide the cleanest coupling in peptidesynthesis. The C-terminal residues of the best segments are eitherdevoid of chiral alpha carbons (glycine or other moieties achiral at thecarbon α to the carboxyl group to be activated in the coupling step) orare compromised of amino acids whose propensity to racemization duringactivation and coupling is lowest of the possible choices. The choice ofN-terminal amino acid for each segment is based on the ease of couplingof an activated acyl intermediate to the amino group. Once the divisionstrategy is selected the method of coupling of each of the segments ischosen based on the synthetic accessibility of the requiredintermediates and the relative ease of manipulation and purification ofthe resulting products (if needed). The segments are then coupledtogether, both in solution, or one on solid phase and the other insolution to prepare the final structure in fully or partially protectedform.

The protected target compound is then subjected to removal of protectinggroups, purified and isolated to give the final desired compound.Advantages of the segment coupling approach are that each segment can bepurified separately, allowing the removal of side products such asdeletion sequences resulting from incomplete couplings or those derivedfrom reactions such as side-chain amide dehydration during couplingsteps, or internal cyclization of side-chains (such as that of Gln) tothe alpha amino group during deprotection of Fmoc groups. Such sideproducts would all be present in the final product of a conventionalresin-based ‘straight through’ peptide chain assembly whereas removal ofthese materials can be performed, if needed, at many stages in a segmentcoupling strategy. Another important advantage of the segment couplingstrategy is that different solvents, reagents and conditions can beapplied to optimize the synthesis of each of the segments to high purityand yield resulting in improved purity and yield of the final product.Other advantages realized are decreased consumption of reagents andlower costs.

EXAMPLES

The following examples are provided as examples of different methodswhich can be used to prepare various compounds of the present invention.Within each example, there are compounds identified in single boldcapital letter (e.g., A, B, C), which correlate to the same labeledcorresponding compounds in the drawings identified.

General Experimental A. Definitions of Abbreviations Used

The following common abbreviations are used throughout thisspecification:

-   1,1-dimethylethoxycarbonyl (Boc or Boc);-   9-fluorenylmethyloxycarbonyl (Fmoc);-   1-hydroxybenozotriazole (HOBt);-   N,N′-diisopropylcarbodiimide (DIC);-   N-methylpyrrolidinone (NMP);-   acetic anhydride (Ac₂O);-   (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (iv-Dde);-   trifluoroacetic acid (TFA);-   Reagent B (TFA:H₂O:phenol:triisopropylsilane, 88:5:5:2);-   diisopropylethylamine (DIEA);-   O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate (HBTU);-   O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorphosphate (HATU);-   N-hydroxysuccinimide (NHS);-   solid phase peptide synthesis (SPPS);-   dimethylsulfoxide (DMSO);-   dichloromethane (DCM);-   dimethylformamide (DMF);-   dimethylacetamide (DMA);-   1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);-   Triisopropylsilane (TIPS);-   1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA)-   (1R)-1-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]ethane-1,2-dicarboxylic    acid (CMDOTA);-   fetal bovine serum (FBS);-   human serum albumin (HSA);-   human prostate cancer cell line (PC3);-   radiochemical purity (RCP); and-   high performance liquid chromatography (HPLC).

B. Materials

The Fmoc-protected amino acids used were purchased from Nova-Biochem(San Diego, Calif., USA), Advanced Chem Tech (Louisville, Ky., USA),Chem-Impex International (Wood Dale Ill., USA), and Multiple PeptideSystems (San Diego, Calif., USA). Other chemicals, reagents andadsorbents required for the syntheses were procured from AldrichChemical Co. (Milwaukee, Wis., USA) and VWR Scientific Products(Bridgeport, N.J., USA). Solvents for peptide synthesis were obtainedfrom Pharmco Co. (Brookfield Conn., USA). Columns for HPLC analysis andpurification were obtained from Waters Co. (Milford, Mass., USA).Experimental details are given below for those that were notcommercially available.

C. Instrumentation for Peptide Synthesis

Peptides were prepared using an Advanced ChemTech 496 ΩMOS synthesizer,an Advanced ChemTech 357 FBS synthesizer and/or by manual peptidesynthesis. However the protocols for iterative deprotection and chainextension employed were the same for all.

D. Instrumentation Employed for Analysis and Purification

Analytical HPLC was performed using a Shimadzu-LC-10A dual pump gradientanalytical LC system employing Shimadzu-ClassVP software version 4.1 forsystem control, data acquisition, and post run processing. Mass spectrawere acquired on a Hewlett-Packard Series 1100 MSD mass spectrometerinterfaced with a Hewlett-Packard Series 1100 dual pump gradient HPLCsystem fitted with an Agilent Technologies 1100 series autosamplerfitted for either direct flow injection or injection onto a WatersAssociates XTerra MS C18 column (4.6 mm×50 mm, 5μ particle, 120 Å pore).The instrument was driven by a HP Kayak workstation using ‘MSD Anyone’software for sample submission and HP Chemstation software forinstrument control and data acquisition. In most cases the samples wereintroduced via direct injection using a 5 μL injection of samplesolution at a concentration of 1 mg/mL and analyzed using positive ionelectrospray to obtain m/e and m/z (multiply charged) ions forconfirmation of structure. ¹H-NMR spectra were obtained on a VarianInnova spectrometer at 500 MHz. ¹³C-NMR spectra were obtained on thesame instrument at 125.73 MHz. Generally the residual ¹H absorption, orin the case of ¹³C-NMR, the ¹³C absorption of the solvent employed, wasused as an internal reference; in other cases tetramethylsilane (6=0.00ppm) was employed. Resonance values are given in 6 units. Micro-analysisdata was obtained from Quantitative Technologies Inc. Whitehouse N.J.Preparative HPLC was performed on a Shimadzu-LC-8A dual pump gradientpreparative HPLC system employing Shimadzu-ClassVP software version 4.3for system control, data acquisition, fraction collection and post runprocessing

E. General Procedure for Peptide Synthesis

Rink Amide-Novagel HL resin (0.6 mmol/g) was used as the solid support.

F. Coupling Procedure

In a typical experiment, the first amino acid was loaded onto 0.1 g ofthe resin (0.06 mmol). The appropriate Fmoc-amino acid in NMP (0.25Msolution; 0.960 mL was added to the resin followed byN-hydroxybenzotriazole (0.5M in NMP; 0.48 mL)) and the reaction block(in the case of automated peptide synthesis) or individual reactionvessel (in the case of manual peptide synthesis) was shaken for about 2min. To the above mixture, diisopropylcarbodiimide (0.5M in NMP; 0.48mL) was added and the reaction mixture was shaken for 4 h at ambienttemperature. Then the reaction block or the individual reaction vesselwas purged of reactants by application of a positive pressure of drynitrogen.

G. Washing Procedure

Each well of the reaction block was filled with 1.2 mL of NMP and theblock was shaken for 5 min. The solution was drained under positivepressure of nitrogen. This procedure was repeated three times. The sameprocedure was used, with an appropriate volume of NMP, in the case ofmanual synthesis using individual vessels.

H. Removal of Fmoc Group

The resin containing the Fmoc-protected amino acid was treated with 1.5mL of 20% piperidine in DMF (v/v) and the reaction block or individualmanual synthesis vessel was shaken for 15 min. The solution was drainedfrom the resin. This procedure was repeated once and the resin waswashed employing the washing procedure described above.

I. Final Coupling of Ligand (DOTA and CMDOTA)

The N-terminal amino group of the resin bound peptide linker constructwas deblocked and the resin was washed. A 0.25M solution of the desiredligand and HBTU in NMP was made, and was treated with a two-foldequivalency of DIEA. The resulting solution of activated ligand wasadded to the resin (1.972 mL; 0.48 mmol) and the reaction mixture wasshaken at ambient temperature for 24-30 h. The solution was drained andthe resin was washed. The final wash of the resin was conducted with 1.5mL dichloromethane (3×).

J. Deprotection and Purification of the Final Peptide

A solution of reagent B (2 mL; 88:5:5:2—TFA:Phenol:Water:TIPS) was addedto the resin and the reaction block or individual vessel was shaken for4.5 h at ambient temperature. The resulting solution containing thedeprotected peptide was drained into a vial. This procedure was repeatedtwo more times with 1 mL of reagent B. The combined filtrate wasconcentrated under reduced pressure using a Genevac HT-12 series IIcentrifugal concentrator. The residue in each vial was then trituratedwith 2 mL of Et₂O and the supernatant was decanted. This procedure wasrepeated twice to provide the peptides as colorless solids. The crudepeptides were dissolved in water/acetonitrile and purified using eithera Waters XTerra MS C18 preparative HPLC column (50 mm×19 mm, 5 micronparticle size, 120 Å pore size) or a Waters-YMC C18 ODS column (250mm×30 mm i.d., 10 micron particle size. 120 Å pore size). The fractionswith the products were collected and analyzed by HPLC. The fractionswith >95% purity were pooled and the peptides isolated bylyophilization.

Conditions for Preparative HPLC (Waters XTerra Column):

-   -   Elution rate: 50 mL/min    -   Detection: UV, λ=220 nm    -   Eluent A: 0.1% aq. TFA; Solvent B: Acetonitrile (0.1% TFA).        Conditions for HPLC Analysis:    -   Column: Waters XTerra (Waters Co.; 4.6×50 mm; MS C18; 5 micron        particle, 120 Å pore).    -   Elution rate: 3 mL/min; Detection: UV, λ=220 nm.    -   Eluent A: 0.1% aq. TFA; Solvent B: Acetonitrile (0.1% TFA).

Example I FIGS. 1A-B Synthesis of L62

Summary: As shown in FIGS. 1A-B, L62 was prepared using the followingsteps: Hydrolysis of (3β,5β)-3-aminocholan-24-oic acid methyl ester Awith NaOH gave the corresponding acid B, which was then reacted withFmoc-Cl to give intermediate C. Rink amide resin functionalised with theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ IDNO:1]) was sequentially reacted with C, Fmoc-glycine and DOTAtri-t-butyl ester. After cleavage and deprotection with reagent B thecrude was purified by preparative HPLC to give L62. Overall yield: 2.5%.More details are provided below:

A. Preparation of Intermediates B and C 1. Synthesis of(3β,5β)-3-Aminocholan-24-oic acid (B)

-   -   A 1 M solution of NaOH (16.6 mL; 16.6 mmol) was added dropwise        to a solution of (3β,5β)-3-aminocholan-24-oic acid methyl ester        (5.0 g; 12.8 mmol) in MeOH (65 mL) at 45° C. After 3 h stirring        at 45° C., the mixture was concentrated to 25 mL and H₂O (40 mL)        and 1 M HCl (22 mL) were added. The precipitated solid was        filtered, washed with H₂O (2×50 mL) and vacuum dried to give B        as a white solid (5.0 g; 13.3 mmol). Yield 80%.

2. Synthesis of (3β,5β)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oicacid (C)

-   -   A solution of 9-fluorenylmethoxycarbonyl chloride (0.76 g; 2.93        mmol) in 1,4-dioxane (9 mL) was added dropwise to a suspension        of (3β,5β)-3-aminocholan-24-oic acid B (1.0 g; 2.66 mmol) in 10%        aq. Na₂CO₃ (16 mL) and 1,4-dioxane (9 mL) stirred at 0° C. After        6 h stirring at room temperature H₂O (90 mL) was added, the        aqueous phase washed with Et₂O (2×90 mL) and then 2 M HCl (15        mL) was added (final pH: 1.5).

The aqueous phase was extracted with EtOAc (2×100 mL), the organic phasedried over Na₂SO₄ and evaporated. The crude was purified by flashchromatography to give C as a white solid (1.2 g; 2.0 mmol). Yield 69%.

B. Synthesis of L62(N-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was shaken for 20 min then the        solution was emptied and the resin washed with DMA (5×7 mL).        (3β,5β)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid C        (0.72 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2        mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol)        and DMA (7 mL) were added to the resin, the mixture shaken for        24 h at room temperature, and the solution was emptied and the        resin washed with DMA (5×7 mL). The resin was then shaken with        50% morpholine in DMA (7 mL) for 10 min, the solution was        emptied, fresh 50% morpholine in DMA (7 mL) was added and the        mixture shaken for another 20 min. The solution was emptied and        the resin washed with DMA (5×7 mL). N-α-Fmoc-glycine (0.79 g;        1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol) and        DMA (7 mL) were added to the resin. The mixture was shaken for 3        h at room temperature, the solution was emptied and the resin        washed with DMA (5×7 mL). The resin was then shaken with 50%        morpholine in DMA (7 mL) for 10 min, the solution was emptied,        fresh 50% morpholine in DMA (7 mL) was added and the mixture        shaken for another 20 min. The solution was emptied and the        resin washed with DMA (5×7 mL) followed by addition of        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) to the resin. The mixture was        shaken for 24 h at room temperature, the solution was emptied        and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and        vacuum dried. The resin was shaken in a flask with reagent B (25        mL) for 4.5 h. The resin was filtered and the solution was        evaporated under reduced pressure to afford an oily crude which        was triturated with Et₂O (20 mL) gave a precipitate. The        precipitate was collected by centrifugation and washed with Et₂O        (3×20 mL), then analysed by HPLC and purified by preparative        HPLC. The fractions containing the product were lyophilised to        give L62 (6.6 mg; 3.8×10⁻³ mmol) as a white solid. Yield 4.5%.

Example II FIGS. 2A-F Synthesis of L70, L73, L74, L115 and L116

Summary: The products were obtained by coupling of the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) (withappropriate side chain protection) on the Rink amide resin withdifferent linkers, followed by functionalization with DOTA tri-t-butylester. After cleavage and deprotection with reagent B the final productswere purified by preparative HPLC. Overall yields 3-9%.

A. Synthesis of L70:

-   -   Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin washed with DMA (5×7 mL).        Fmoc-4-aminobenzoic acid (0.43 g; 1.2 mmol), HOBt (0.18 g; 1.2        mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the        resin, the mixture shaken for 3 h at room temperature, the        solution was emptied and the resin washed with DMA (5×7 mL). The        resin was then shaken with 50% morpholine in DMA (7 mL) for 10        min, the solution was emptied, fresh 50% morpholine in DMA (7        mL) was added and the mixture was shaken for 20 min. The        solution was emptied and the resin washed with DMA (5×7 mL).        Fmoc-glycine (0.36 g; 1.2 mmol),        N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate (HATU) (0.46 g; 1.2 mmol) and DIEA (0.40 mL;        2.4 mmol) were stirred for 15 min in DMA (7 mL) then the        solution was added to the resin, the mixture shaken for 2 h at        room temperature, the solution was emptied and the resin washed        with DMA (5×7 mL). The resin was then shaken with 50% morpholine        in DMA (7 mL) for 10 min, the solution was emptied, fresh 50%        morpholine in DMA (7 mL) was added and the mixture shaken for 20        min. The solution was emptied and the resin washed with DMA (5×7        mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7        mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        filtrate solution was evaporated under reduced pressure to        afford an oily crude that was triturated with Et₂O (5 mL). The        precipitate was collected by centrifugation and washed with Et₂O        (5×5 mL), then analysed by HPLC and purified by preparative        HPLC. The fractions containing the product were lyophilised to        give L70 as a white fluffy solid (6.8 mg; 0.005 mmol). Yield 3%.

B. Synthesis of L73 L115 and L116:

-   -   Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin washed with DMA (5×7 mL).        Fmoc-linker (1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g;        1.2 mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2        mmol) and DMA (7 mL) were added to the resin, the mixture was        shaken for 3 h at room temperature, the solution was emptied and        the resin was washed with DMA (5×7 mL). The resin was shaken        with 50% morpholine in DMA (7 mL) for 10 min, the solution was        emptied, fresh 50% morpholine in DMA (7 mL) was added and the        mixture was shaken for 20 min. The solution was emptied and the        resin washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7        mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        solution was evaporated under reduced pressure to afford an oily        crude that was triturated with Et₂O (5 mL). The precipitate was        collected by centrifugation and washed with Et₂O (5×5 mL), then        analysed by HPLC and purified by preparative HPLC. The fractions        containing the product were lyophilised.

D. Synthesis of L74:

-   -   Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin was washed with DMA (5×7 mL).        Fmoc-isonipecotic acid (0.42 g; 1.2 mmol), HOBt (0.18 g; 1.2        mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the        resin, the mixture was shaken for 3 h at room temperature, the        solution was emptied and the resin was washed with DMA (5×7 mL).        The resin was shaken with 50% morpholine in DMA (7 mL) for 10        min, the solution was emptied, fresh 50% morpholine in DMA (7        mL) was added and the mixture was shaken for 20 min. The        solution was emptied and the resin was washed with DMA (5×7 mL).        Fmoc-glycine (0.36 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC        (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the        mixture was shaken for 3 h at room temperature, the solution was        emptied and the resin washed with DMA (5×7 mL). The resin was        then shaken with 50% morpholine in DMA (7 mL) for 10 min, the        solution was emptied, fresh 50% morpholine in DMA (7 mL) was        added and the mixture shaken for 20 minutes. The solution was        emptied and the resin was washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin was washed with DMA (5×7 mL), CH₂Cl₂        (5×7 mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        solution was evaporated under reduced pressure to afford an oily        crude that was triturated with Et₂O (5 mL). The precipitate was        collected by centrifugation and washed with Et₂O (5×5 mL), then        analysed by HPLC and purified by HPPLC. The fractions containing        the product were lyophilised to give L74 as a white fluffy solid        (18.0 mg; 0.012 mmol). Yield 8%.

Example III FIGS. 3A-E Synthesis of L67

Summary: Hydrolysis of (3β,5β)-3-amino-12-oxocholan-24-oic acid methylester A with NaOH gave the corresponding acid B, which was then reactedwith Fmoc-Glycine to give intermediate C. Rink amide resinfunctionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂(BBN[7-14] [SEQ ID NO:1]) was sequentially reacted with C, and DOTAtri-t-butyl ester. After cleavage and deprotection with reagent B thecrude was purified by preparative HPLC to give L67. Overall yield: 5.2%.

A. Synthesis (3β,5β)-3-Amino-12-oxocholan-24-oic acid, (B)

-   -   A 1 M solution of NaOH (6.6 mL; 6.6 mmol) was added dropwise to        a solution of (3β,5β)-3-amino-12-oxocholan-24-oic acid methyl        ester A (2.1 g; 5.1 mmol) in MeOH (15 mL) at 45° C. After 3 h        stirring at 45° C., the mixture was concentrated to 25 mL then        H₂O (25 mL) and 1 M HCl (8 mL) were added. The precipitated        solid was filtered, washed with H₂O (2×30 mL) and vacuum dried        to give B as a white solid (1.7 g; 4.4 mmol). Yield 88%.

B. Synthesis of(3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxocholan-24-oicacid (C)

-   -   Tributylamine (0.7 mL; 3.1 mmol) was added dropwise to a        solution of N-α-Fmoc-glycine (0.9 g; 3.1 mmol) in THF (25 mL)        stirred at 0° C. Isobutyl chloroformate (0.4 mL; 3.1 mmol) was        subsequently added and, after 10 min, a suspension of        tributylamine (0.6 mL; 2.6 mmol) and        (3β,5β)-3-amino-12-oxocholan-24-oic acid B (1.0 g; 2.6 mmol) in        DMF (30 mL) was added dropwise, over 1 h, into the cooled        solution. The mixture was allowed to warm up and after 6 h the        solution was concentrated to 40 mL, then H₂O (50 mL) and 1 N HCl        (10 mL) were added (final pH: 1.5). The precipitated solid was        filtered, washed with H₂O (2×50 mL), vacuum dried and purified        by flash chromatography to give C as a white solid (1.1 g; 1.7        mmol). Yield 66%.

C. Synthesis of L67(N-[(3β,5β)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin was washed with DMA (5×7 mL).        (3β,5β)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino]-12-oxocholan-24-oic        acid C (0.80 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18        g; 1.2 mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2        mmol) and DMA (7 mL) were added to the resin, the mixture was        shaken for 24 h at room temperature, the solution was emptied        and the resin was washed with DMA (5×7 mL). The resin was shaken        with 50% morpholine in DMA (7 mL) for 10 min, the solution was        emptied, fresh 50% morpholine in DMA (7 mL) was added and the        mixture was shaken for 20 min. The solution was emptied and the        resin was washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin was washed with DMA (5×7 mL), CH₂Cl₂        (5×7 mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4.5 h. The resin was filtered and the        solution was evaporated under reduced pressure to afford an oily        crude that was triturated with Et₂O (20 mL).

Example IV FIGS. 4A-H Synthesis of L63 and L64

Summary: Hydrolysis of(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid methyl ester 1bwith NaOH gave the intermediate 2b, which was then reacted withFmoc-glycine to give 3b. Rink amide resin functionalised with theoctapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ IDNO:1]) was reacted with 3b and then with DOTA tri-t-butyl ester. Aftercleavage and deprotection with reagent B the crude was purified bypreparative HPLC to give L64. The same procedure was repeated startingfrom intermediate 2a, already available, to give L63. Overall yields: 9and 4%, respectively.

A. Synthesis of (3β,5β,7α,12α)-3-Amino-7,12-dihydroxycholan-24-oic acid,(2b)

-   -   A 1 M solution of NaOH (130 mL; 0.13 mol) was added dropwise to        a solution of (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic        acid methyl ester 1b (42.1 g; 0.10 mol) in MeOH (300 mL) heated        at 45° C. After 3 h stirring at 45° C., the mixture was        concentrated to 150 mL and H₂O (350 mL) was added. After        extraction with CH₂Cl₂ (2×100 mL) the aqueous solution was        concentrated to 200 mL and 1 M HCl (150 mL) was added. The        precipitated solid was filtered, washed with H₂O (2×100 mL) and        vacuum dried to give 2b as a white solid (34.8 g; 0.08 mol).        Yield 80%.

B. Synthesis of(3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oicacid, (3a)

-   -   Tributylamine (4.8 mL; 20.2 mmol) was added dropwise to a        solution of N-α-Fmoc-glycine (6.0 g; 20.2 mmol) in THF (120 mL)        stirred at 0° C. Isobutyl chloroformate (2.6 mL; 20.2 mmol) was        subsequently added and, after 10 min, a suspension of        tributylamine (3.9 mL; 16.8 mmol) and        (3β,5β,12α)-3-amino-12-hydroxycholan-24-oic acid 2a (6.6 g; 16.8        mmol) in DMF (120 mL) was added dropwise, over 1 h, into the        cooled solution. The mixture was allowed to warm up and after 6        h the solution was concentrated to 150 mL, then H₂O (250 mL) and        1 N HCl (40 mL) were added (final pH: 1.5). The precipitated        solid was filtered, washed with H₂O (2×100 mL), vacuum dried and        purified by flash chromatography to give 3a as a white solid        (3.5 g; 5.2 mmol). Yield 31%

C. Synthesis of(3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid, (3b)

-   -   Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a        solution of N-α-Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL)        stirred at 0° C. Isobutyl chloroformate (1.7 mL; 13.5 mmol) was        subsequently added and, after 10 min, a suspension of        tributylamine (2.6 mL; 11.2 mmol) and        (3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid 3a (4.5        g; 11.2 mmol) in DMF (80 mL) was added dropwise, over 1 h, into        the cooled solution. The mixture was allowed to warm up and        after 6 h the solution was concentrated to 120 mL, then H₂O (180        mL) and 1 N HCl (30 mL) were added (final pH: 1.5). The        precipitated solid was filtered, washed with H₂O (2×100 mL),        vacuum dried and purified by flash chromatography to give 3a as        a white solid (1.9 g; 2.8 mmol). Yield 25%. In an alternative        method,        (3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic        acid, (3b) can be prepared as follows:        -   N-Hydroxysuccinimide (1.70 g, 14.77 mmol) and DIC (1.87 g,            14.77 mmol) were added sequentially to a stirred solution of            Fmoc-Gly-OH (4.0 g, 13.45 mmol) in dichloromethane (15 mL);            the resulting mixture was stirred at room temperature for            4 h. The N,N′-diisopropylurea formed was removed by            filtration and the solid was washed with ether (20 mL). The            volatiles were removed and the solid Fmoc-Gly-succinimidyl            ester formed was washed with ether (3×20 mL).            Fmoc-Gly-succinimidyl ester was then redissolved in dry DMF            (15 mL) and 3-aminodeoxycholic acid (5.21 g, 12.78 mmol) was            added to the clear solution. The reaction mixture was            stirred at room temperature for 4 h, water (200 mL) was            added and the precipitated solid was filtered, washed with            water, dried and purified by silica gel chromatography (TLC            (silica): (R_(f): 0.50, silica gel, CH₂Cl₂/CH₃OH, 9:1)            (eluant: CH₂Cl₂/CH₃OH (9:1) to give Fmoc-Gly-3-aminocholic            acid as a colorless solid. Yield: 7.46 g (85%).

D. Synthesis of L63(N-[(3β,5β,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin washed with DMA (5×7 mL).        (3β,5β,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic        acid 3a (0.82 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18        g; 1.2 mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2        mmol) and DMA (7 mL) were added to the resin, the mixture was        shaken for 24 h at room temperature, the solution was emptied        and the resin was washed with DMA (5×7 mL). The resin was then        shaken with 50% morpholine in DMA (7 mL) for 10 min, the        solution was emptied, fresh 50% morpholine in DMA (7 mL) was        added and the mixture was shaken for 20 min. The solution was        emptied and the resin washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7        mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        solution was evaporated under reduced pressure to afford an oily        crude that after treatment with Et₂O (5 mL) gave a precipitate.        The precipitate was collected by centrifugation and washed with        Et₂O (5×5 mL), then analysed and purified by HPLC. The fractions        containing the product were lyophilised to give L63 as a white        fluffy solid (12.8 mg; 0.0073 mmol). Yield 9%.

E. Synthesis of L64(N-[(3β,5β,7α,12α)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min, the solution        was emptied and the resin was washed with DMA (5×7 mL).        (3β,5β,7α,12α)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic        acid 3b (0.81 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19        mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin was washed with DMA (5×7 mL). The        resin was shaken with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied, fresh 50% morpholine in DMA (7 mL) was        added and the mixture was shaken for 20 min. The solution was        emptied and the resin was washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7        mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        solution was evaporated under reduced pressure to afford an oily        crude that was triturated with Et₂O (5 mL). The precipitate was        collected by centrifugation and washed with Et₂O (5×5 mL). Then        it was dissolved in H₂O (20 mL), and Na₂CO₃ (0.10 g; 0.70 mmol)        was added; the resulting mixture was stirred 4 h at room        temperature. This solution was purified by HPLC, the fractions        containing the product lyophilised to give L64 as a white fluffy        solid (3.6 mg; 0.0021 mmol). Yield 4%.

Example V FIGS. 5A-E Synthesis of L71 and L72

Summary: The products were obtained in two steps. The first step was thesolid phase synthesis of the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) (withappropriate side chain protecting groups) on the Rink amide resin. Thesecond step was the coupling with different linkers followed byfunctionalization with DOTA tri-t-butyl ester. After cleavage anddeprotection with reagent B the final products were purified bypreparative HPLC. Overall yields 3-9%.

A. Rink Amide Resin Functionalised with bombesin[7-14], (B)

-   -   In a solid phase peptide synthesis vessel (see enclosure No. 1)        Fmoc-amino acid (24 mmol), N-hydroxybenzotriazole (HOBt) (3.67        g; 24 mmol), and N,N′-diisopropylcarbodiimide (DIC) (3.75 mL; 24        mmol) were added sequentially to a suspension of Rink amide        NovaGel™ resin (10 g; 6.0 mmol) A in DMF (45 mL). The mixture        was shaken for 3 h at room temperature using a bench top shaker,        then the solution was emptied and the resin was washed with DMF        (5×45 mL). The resin was shaken with 25% piperidine in DMF (45        mL) for 4 min, the solution was emptied and fresh 25% piperidine        in DMF (45 mL) was added. The suspension was shaken for 10 min,        then the solution was emptied and the resin was washed with DMF        (5×45 mL).    -   This procedure was applied sequentially for the following amino        acids: N-α-Fmoc-L-methionine, N-α-Fmoc-L-leucine,        N-α-Fmoc-N-im-trityl-L-histidine, N-α-Fmoc-glycine,        N-α-Fmoc-L-valine, N-α-Fmoc-L-alanine,        N-α-Fmoc-N-in-Boc-L-tryptophan.    -   In the last coupling reaction N-α-Fmoc-N-γ-trityl-L-glutamine        (14.6 g; 24 mmol), HOBt (3.67 g; 24 mmol), and DIC (3.75 mL; 24        mmol) were added to the resin in DMF (45 mL). The mixture was        shaken for 3 h at room temperature, the solution was emptied and        the resin was washed with DMF (5×45 mL), CH₂Cl₂ (5×45 mL) and        vacuum dried.

B. Bombesin [7-14] Functionalisation and Cleavage Procedure

-   -   The resin B (0.5 g; 0.3 mmol) was shaken in a solid phase        peptide synthesis vessel with 50% morpholine in DMA (7 mL) for        10 min, the solution was emptied and fresh 50% morpholine in DMA        (7 mL) was added. The suspension was stirred for 20 min then the        solution was emptied and the resin was washed with DMA (5×7 mL).        The Fmoc-linker (1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19        mL; 1.2 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 3 h at room temperature, the solution was        emptied and the resin washed with DMA (5×7 mL). The resin was        then shaken with 50% morpholine in DMA (7 mL) for 10 min, the        solution was emptied, fresh 50% morpholine in DMA (7 mL) was        added and the mixture was shaken for 20 min. The solution was        emptied and the resin was washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl C (0.79 g; 1.2        mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA        (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature. The solution        was emptied and the resin washed with DMA (5×7 mL), CH₂Cl₂ (5×7        mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        filtrate was evaporated under reduced pressure to afford an oily        crude that was triturated with ether (5 mL). The precipitate was        collected by centrifugation and washed with ether (5×5 mL), then        analyzed by analytical HPLC and purified by preparative HPLC.        The fractions containing the product were lyophilized.

C. Products 1. L71(4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycil-L-histidyl-L-leucyl-L-methioninamide)

-   -   The product was obtained as a white fluffy solid (7.3 mg; 0.005        mmol). Yield 7.5%.

2. L72(Trans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]cyclohexylcarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycil-L-histidyl-L-leucyl-L-methioninamide)

-   -   The product was obtained as a white fluffy solid (7.0 mg; 0.005        mmol). Yield 4.8%.

D.Trans-4-[[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylicacid, (D)

-   -   A solution of N-(9-fluorenylmethoxycarbonyloxy)succinimide (4.4        g; 14.0 mmol) in 1,4-dioxane (40 mL) was added dropwise to a        solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (2.0        g; 12.7 mmol) in 10% Na₂CO₃ (30 mL) cooled to 0° C. The mixture        was then allowed to warm to ambient temperature and after 1 h        stirring at room temperature was treated with 1 N HCl (32 mL)        until the final pH was 2. The resulting solution was extracted        with n-BuOH (100 mL); the volatiles were removed and the crude        residue was purified by flash chromatography to give D as a        white solid (1.6 g; 4.2 mmol). Yield 33%.

Example VI FIGS. 6A-K Synthesis of L75 and L76

Summary: The two products were obtained by coupling of the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) on theRink amide resin with the two linkers E and H, followed byfunctionalization with DOTA tri-t-butyl ester. After cleavage anddeprotection with reagent B the final products were purified bypreparative HPLC. Overall yields: 8.5% (L75) and 5.6% (L76).

A. 2-[(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid, (C)

-   -   The product was synthesized following the procedure reported in        the literature (Bornstein, J; Drummon, P. E.; Bedell, S. F. Org.        Synth. Coll. Vol. IV 1963, 810-812).

B. 2-(Aminomethyl)benzoic acid, (D)

-   -   A 40% solution of methylamine (6.14 mL; 7.1 mmol) was added to        2-[(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl]benzoic acid C        (2 g; 7.1 mmol) and then EtOH (30 mL) was added. After 5 minutes        stirring at room temperature the reaction mixture was heated at        50° C. After 2.5 h, the mixture was cooled and the solvent was        evaporated under reduced pressure. The crude product was        suspended in 50 mL of absolute ethanol and the suspension was        stirred at room temperature for 1 h. The solid was filtered and        washed with EtOH to afford 2-(aminomethyl)benzoic acid D (0.87        g; 5.8 mmol). Yield 81%.

C. 2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid, (E)

-   -   The product was synthesized following the procedure reported in        the literature (Sun, J-H.; Deneker, W. F. Synth. Commun. 1998,        28, 4525-4530).

D. 4-(Aminomethyl)-3-nitrobenzoic acid, (G)

-   -   4-(Bromomethyl)-3-nitrobenzoic acid (3.2 g; 12.3 mmol) was        dissolved in 8% NH₃ in EtOH (300 mL) and the resulting solution        was stirred at room temperature. After 22 h the solution was        evaporated and the residue suspended in H₂O (70 mL). The        suspension was stirred for 15 min and filtered. The collected        solid was suspended in H₂O (40 mL) and dissolved by the addition        of few drops of 25% aq. NH₄OH (pH 12), then the pH of the        solution was adjusted to 6 by addition of 6 N HCl. The        precipitated solid was filtered, and washed sequentially with        MeOH (3×5 mL), and Et₂O (10 mL) and was vacuum dried (1.3 kPa;        P₂O₅) to give 4-(aminomethyl)-3-nitrobenzoic acid as a pale        brown solid (1.65 g; 8.4 mmol). Yield 68%.

E. 4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoicacid, (H)

-   -   4-(Aminomethyl)-3-nitrobenzoic acid G (0.8 g; 4 mmol) was        dissolved in 10% aq. Na₂CO₃ (25 mL) and 1,4-dioxane (10 mL) and        the solution was cooled to 0° C. A solution of 9-fluorenylmethyl        chloroformate (Fmoc-Cl) (1.06 g; 4 mmol) in 1,4-dioxane (10 mL)        was added dropwise for 20 min. After 2 h at 0-5° C. and 1 h at        10° C. the reaction mixture was filtered and the solution was        acidified to pH 5 by addition of 1 N HCl. The precipitate was        filtered, washed with H₂O (2×2 mL) dried under vacuum (1.3 kPa;        P₂O₅) to give        4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic        acid as a white solid (1.6 g; 3.7 mmol). Yield 92%.

F. L75(N-[2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin I (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin washed with DMA (5×7 mL).        2-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]benzoic acid,        E (0.45 g; 1.2 mmol), N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2        mmol), N,N′-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol)        and DMA (7 mL) were added to the resin, the mixture shaken for        24 h at room temperature, the solution was emptied and the resin        was washed with DMA (5×7 mL). The resin was then shaken with 50%        morpholine in DMA (7 mL) for 10 min, the solution was emptied,        fresh 50% morpholine in DMA (7 mL) was added and the mixture        shaken for 20 min. The solution was emptied and the resin washed        with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (DOTA tri-t-butyl        ester) (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19        mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were        added to the resin. The mixture was shaken for 24 h at room        temperature, the solution was emptied and the resin was washed        with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The resin        was shaken in a flask with reagent B (25 mL) for 4.5 h. The        resin was filtered and the filtrate was evaporated under reduced        pressure to afford an oily crude that after treatment with Et₂O        (20 mL) gave a precipitate. The resulting precipitate was        collected by centrifugation and was washed with Et₂O (3×20 mL)        to give L75 (190 mg; 0.13 mmol) as a white solid. Yield 44%.

G. L76(N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin I (0.5 g; 0.3 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin was washed with DMA (5×7 mL).        4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic        acid, H (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19        mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin was washed with DMA (5×7 mL). The        resin was then shaken with 50% morpholine in DMA (7 mL) for 10        min, the solution was emptied, fresh 50% morpholine in DMA (7        mL) was added and the mixture was shaken for 20 min. The        solution was emptied and the resin was washed with DMA (5×7 mL).        DOTA tri-t-butyl ester (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2        mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA        (7 mL) were added to the resin. The mixture was shaken for 24 h        at room temperature, the solution was emptied and the resin was        washed with DMA (5×7 mL), CH₂Cl₂ (5×7 mL) and vacuum dried. The        resin was shaken in a flask with reagent B (25 mL) for 4.5 h.        The resin was filtered and the solution was evaporated under        reduced pressure to afford an oily crude that was triturated        with Et₂O (20 mL). The precipitate was collected by        centrifugation and was washed with Et₂O (3×20 mL) to give a        solid (141 mg) which was analysed by HPLC. A 37 mg portion of        the crude was purified by preparative HPLC. The fractions        containing the product were lyophilised to give L76 (10.8 mg;        7.2×10⁻³ mmol) as a white solid. Yield 9%.

Example VII FIGS. 7A-F Synthesis of L124

Summary: 4-Cyanophenol A was reacted with ethyl bromoacetate and K₂CO₃in acetone to give the intermediated B, which was hydrolysed with NaOHto the corresponding acid C. Successive hydrogenation of C with H₂ andPtO₂ at 355 kPa in EtOH/CHCl₃ gave the corresponding amino acid D, whichwas directly protected with FmocOSu to give E. Rink amide resinfunctionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂(BBN[7-14] [SEQ ID NO:1]) was reacted with E and then with DOTAtri-t-butyl ester. After cleavage and deprotection with reagent B thecrude was purified by preparative HPLC to give L124. Overall yield: 1.3%

A. Synthesis of (4-Cyanophenoxy)acetic acid ethyl ester, (B)

-   -   The product was synthesized following the procedure reported in        the literature (Archimbault, P.; LeClerc, G.; Strosberg, A. D.;        Pietri-Rouxel, F. PCT Int. Appl. WO 980005, 1998).

B. Synthesis of (4-Cyanophenoxy)acetic acid, (C)

-   -   A 1 N solution of NaOH (7.6 mL; 7.6 mmol) was added dropwise to        a solution of (4-cyanophenoxy)acetic acid ethyl ester B (1.55 g;        7.6 mmol) in MeOH (15 mL). After 1 h the solution was acidified        with 1 N HCl (7.6 mL; 7.6 mmol) and evaporated. The residue was        taken up with water (20 mL) and extracted with CHCl₃ (2×30 mL).        The organic phases were evaporated and the crude was purified by        flash chromatography to give (4-cyanophenoxy)acetic acid C (0.97        g; 5.5 mmol) as a white solid. Yield 72%.

C. Synthesis of[4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid,(E)

-   -   PtO₂ (150 mg) was added to a solution of (4-cyanophenoxy)acetic        acid C (1.05 g; 5.9 mmol) in EtOH (147 mL) and CHCl₃ (3 mL). The        suspension was stirred 30 h under a hydrogen atmosphere (355        kPa; 20° C.). The mixture was filtered through a Celite® pad and        the solution evaporated under vacuum. The residue was purified        by flash chromatography to give acid D (0.7 g) which was        dissolved in H₂O (10 mL), MeCN (2 mL) and Et₃N (0.6 mL) at 0°        C., then a solution of        N-(9-fluorenylmethoxycarbonyloxy)succinimide (1.3 g; 3.9 mmol)        in MeCN (22 mL) was added dropwise. After stirring 16 h at room        temperature the reaction mixture was filtered and the volatiles        were removed under vacuum. The residue was treated with 1 N HCl        (10 mL) and the precipitated solid was filtered and purified by        flash chromatography to give        [4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic        acid E (0.56 g; 1.4 mmol) as a white solid. Overall yield 24%.

E. Synthesis of L124(N-[[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]phenoxy]acetyl]-L-glutaminyl-L-trytophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin F (480 mg; 0.29 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min, the solution        was emptied and the resin was washed with DMA (5×7 mL).        [4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic        acid E (480 mg; 1.19 mmol), N-hydroxybenzotriazole (HOBt) (182        mg; 1.19 mmol), N,N′-diisopropylcarbodiimide (DIC) (185 μL; 1.19        mmol) and DMA (7 mL) were added to the resin, the mixture was        shaken for 24 h at room temperature, the solution was emptied        and the resin was washed with DMA (5×7 mL). The resin was then        shaken with 50% morpholine in DMA (6 mL) for 10 min, the        solution was emptied, fresh 50% morpholine in DMA (6 mL) was        added and the mixture was shaken for 20 min. The solution was        emptied and the resin was washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (750 mg; 1.19        mmol), HOBt (182 mg; 1.19 mmol), DIEA (404 μL; 2.36 mmol), DIC        (185 μL; 1.19 mmol) and DMA (6 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied, the resin was washed with DMA (2×7 mL), CH₂Cl₂ (5×7        mL) and vacuum dried. The resin was shaken in a flask with        Reagent B (25 mL) for 4 h. The resin was filtered and the        filtrate was evaporated under reduced pressure to afford an oily        crude that was triturated with Et₂O (5 mL). The precipitate was        collected by centrifugation and washed with Et₂O (5×5 mL) to        give a solid (148 mg) which was analysed by HPLC. A 65 mg        portion of the crude was purified by preparative HPLC. The        fractions containing the product were lyophilised to give L127        as a white solid (15 mg; 0.01 mmol). Yield 7.9%.

Example VIII FIGS. 8A-F Synthesis of L125

Summary: 4-(Bromomethyl)-3-methoxybenzoic acid methyl ester A wasreacted with NaN₃ in DMF to give the intermediate azide B, which wasthen reduced with Ph₃P and H₂O to amine C. Hydrolysis of C with NaOHgave acid D, which was directly protected with FmocOSu to give E. Rinkamide resin functionalised with the octapeptideGln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (BBN[7-14] [SEQ ID NO:1]) wasreacted with E and then with DOTA tri-t-butyl ester. After cleavage anddeprotection with reagent B the crude was purified by preparative HPLCto give L125. Overall yield: 0.2%.

A. Synthesis of 4-(Azidomethyl)-3-methoxybenzoic acid methyl ester, (B)

-   -   A solution of 4-(bromomethyl)-3-methoxybenzoic acid methyl ester        (8 g; 31 mmol) and NaN₃(2 g; 31 mmol) in DMF (90 mL) was stirred        overnight at room temperature. The volatiles were removed under        vacuum and the crude product was dissolved in EtOAc (50 mL). The        solution was washed with water (2×50 mL) and dried. The        volatiles were evaporated to provide        4-(azidomethyl)-3-methoxybenzoic acid methyl ester (6.68 g; 30        mmol). Yield 97%.

B. 4-(Aminomethyl)-3-methoxybenzoic acid methyl ester, (C)

-   -   Triphenylphosphine (6.06 g; 23 mmol) was added to a solution of        (4-azidomethyl)-3-methoxybenzoic acid methyl ester B (5 g; 22        mmol) in THF (50 mL): hydrogen evolution and formation of a        white solid was observed. The mixture was stirred under nitrogen        at room temperature. After 24 h more triphenylphosphine (0.6 g;        2.3 mmol) was added. After 24 h the azide was consumed and H₂O        (10 mL) was added. After 4 h the white solid disappeared. The        mixture was heated at 45° C. for 3 h and was stirred overnight        at room temperature. The solution was evaporated to dryness and        the crude was purified by flash chromatography to give        4-(aminomethyl)-3-methoxybenzoic acid methyl ester C (1.2 g; 6.1        mmol). Yield 28%.

C. 4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoicacid, (E)

-   -   A 1 N solution of NaOH (6.15 mL; 6.14 mmol) was added dropwise        to a solution of 4-(aminomethyl)-3-methoxybenzoic acid methyl        ester C (1.2 g; 6.14 mmol) in MeOH (25 mL) heated at 40° C.        After stirring 8 h at 45° C. the solution was stirred over night        at room temperature. A 1 N solution of NaOH (0.6 mL; 0.6 mmol)        was added and the mixture heated at 40° C. for 4 h. The solution        was concentrated, acidified with 1 N HCl (8 mL; 8 mmol),        extracted with EtOAc (2×10 mL) then the aqueous layer was        concentrated to 15 mL. This solution (pH 4.5) was cooled at        0° C. and Et₃N (936 μL; 6.75 mmol) was added (pH 11). A solution        of N-(9-fluorenylmethoxycarbonyloxy)succinimide (3.04 g; 9 mmol)        in MeCN (30 mL) was added dropwise (final pH 9) and a white        solid precipitated. After stirring 1 h at room temperature the        solid was filtered, suspended in 1N HCl (15 mL) and the        suspension was stirred for 30 min. The solid was filtered to        provide        4-[[[9H-fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic        acid E as a white solid (275 mg; 0.7 mmol).    -   The filtrate was evaporated under vacuum and the resulting white        residue was suspended in 1N HCl (20 mL) and stirred for 30        minutes. The solid was filtered and purified by flash        chromatography to give more acid E (198 mg; 0.5 mmol). Overall        yield 20%.

D. L125(N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)

-   -   Resin F (410 mg; 0.24 mmol) was shaken in a solid phase peptide        synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min,        the solution was emptied and fresh 50% morpholine in DMA (7 mL)        was added. The suspension was stirred for 20 min then the        solution was emptied and the resin was washed with DMA (5×7 mL).        4-[[[9H-Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic        acid E (398 mg; 0.98 mmol), N-hydroxybenzotriazole (HOBt) (151        mg; 0.98 mmol), N,N′-diisopropylcarbodiimide (DIC) (154 μL; 0.98        mmol) and DMA (6 mL) were added to the resin; the mixture was        shaken for 24 h at room temperature, the solution was emptied        and the resin was washed with DMA (5×7 mL). The resin was then        shaken with 50% morpholine in DMA (6 mL) for 10 min, the        solution was emptied, fresh 50% morpholine in DMA (6 mL) was        added and the mixture was shaken for 20 min. The solution was        emptied and the resin washed with DMA (5×7 mL).        1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid        tris(1,1-dimethylethyl) ester adduct with NaCl (618 mg; 0.98        mmol), HOBt (151 mg; 0.98 mmol), DIC (154 μL; 0.98 mmol), DIEA        (333 μL; 1.96 mmol) and DMA (6 mL) were added to the resin. The        mixture was shaken for 24 h at room temperature, the solution        was emptied and the resin was washed with DMA (5×7 mL), CH₂Cl₂        (5×7 mL) and vacuum dried. The resin was shaken in a flask with        reagent B (25 mL) for 4 h. The resin was filtered and the        solution was evaporated under reduced pressure to afford an oily        crude that was triturated with Et₂O (5 mL). The resulting        precipitate was collected by centrifugation, was washed with        Et₂O (5×5 mL), was analysed by HPLC and purified by preparative        HPLC. The fractions containing the product were lyophilised to        give L125 as a white solid (15.8 mg; 0.011 mmol). Yield 4.4%.

Example IX Synthesis of Additional GRP Compounds A. General procedurefor the preparation of 4,4′-Aminomethylbiphenylcarboxylic acid (B2) and3,3′-aminomethylbiphenylcarboxylic acid (B3) 1Methyl-hydroxymethylbiphenylcarboxylates

-   -   Commercially available (Aldrich Chemical Co.)        4-hydroxymethylphenylboric acid or 3-hydroxymethylphenylboric        acid (1.0 g, 6.58 mmol) was stirred with isopropanol (10 mL) and        2M sodium carbonate (16 mL) until the solution became        homogeneous. The solution was degassed by passing nitrogen        through the solution and then treated with solid        methyl-3-bromobenzoate, or methyl-4-bromobenzoate (1.35 g, 6.3        mmol) followed by the Pd (0) catalyst {[(C₆H₅)₃P]₄Pd; 0.023 g,        0.003 mmol}. The reaction mixture was kept at reflux under        nitrogen until the starting bromobenzoate was consumed as        determined by TLC analysis (2-3 h). The reaction mixture was        then diluted with 250 mL of water and extracted with ethyl        acetate (3×50 mL). The organic layers were combined and washed        with saturated sodium bicarbonate solution (2×50 mL) and dried        (Na₂SO₄). The solvent was removed under reduced pressure and the        residue was chromatographed over flash silica gel (100 g).        Elution with 40% ethyl acetate in hexanes yielded the product        either as a solid or oil.        Yield:

B2—0.45 g (31%); m. p.—170-171° C.

B3—0.69 g (62%); oil.

¹H NMR (CDCl₃) δ B2—3.94 (s, 3H, —COOCH₃), 4.73 (s, 2H, —CH₂-Ph), 7.475(d, 2H, J=5 Hz), 7.6 (d, 2H, J=10 Hz), 7.65 (d, 2H, J=5 Hz) and 8.09 (d,2H, J=10 Hz).

M. S.—m/e—243.0 [M+H]

B3—3.94 (s, 3H, —COOCH₃), 4.76 (s, 2H, —CH₂-Ph), 7.50 (m, 4H), 7.62 (s,1H), 7.77 (s, 1H), 8.00 (s, 1H) and 8.27 (s, 1H).

M. S.—m/e—243.2 [M+H]

2. Azidomethylbiphenyl Carboxylates

-   -   The above biphenyl alcohols (2.0 mmol) in dry dichloromethane        (10 mL) were cooled in ice and treated with diphenylphosphoryl        azide (2.2 mol) and DBU (2.0 mmol) and stirred under nitrogen        for 24 h. The reaction mixture was diluted with water and        extracted with ethyl acetate (2×25 mL). The organic layers were        combined and washed successfully with 0.5 M citric acid solution        (2×25 mL), water (2×25 mL) and dried (Na₂SO₄). The solution was        filtered and evaporated under reduced pressure to yield the        crude product. The 4,4′-isomer was crystallized from        hexane/ether and the 3,3′-isomer was triturated with isopropyl        ether to remove all the impurities; the product was homogeneous        as determined on TLC analysis and further purification was not        required.        Yield:

Methyl-4-azidomethyl-4-biphenylcaroxylate—0.245 g (46%); m. p.—106-108°C.

Methyl-4-azidomethyl-4-biphenylcaroxylate—0.36 g (59%, oil)

¹H NMR (CDCl₃) δ—4,4′-isomer-3.95 (s, 3H, —COOCH₃), 4.41 (s, 2H,—CH₂N₃), 7.42 (d, 2H, J=5 Hz), 7.66 (m, 4H) and 8.11 (d, 2H, J=5 Hz)

3,3′-Isomer—3.94 (s, 3H, —COOCH₃), 4.41 (s, 2H, —CH₂N₃), 7.26-7.6 (m,5H), 7.76 (d, 1H, J=10 Hz), 8.02 (d, 1H, J=5 Hz) and 8.27 (s, 1H).

3. Hydrolysis of the Methyl Esters of Biphenylcarboxylates

-   -   About 4 mmol of the methyl esters were treated with 20 mL of 2M        lithium hydroxide solution and stirred until the solution was        homogeneous (20-24 h). The aqueous layer was extracted with 2×50        mL of ether and the organic layer was discarded. The aqueous        layer was then acidified with 0.5 M citric acid and the        precipitated solid was filtered and dried. No other purification        was necessary and the acids were taken to the next step.        Yield:

4,4′-isomer—0.87 g of methyl ester yielded 0.754 g of the acid (86.6%);m. p.—205-210° C.

3,3′-isomer—0.48 g of the methyl ester furnished 0.34 g of the acid(63.6%); m. p.—102-105° C.

¹H NMR (DMSO-d₆) δ: 4,4′-isomer—4.52 (s, 2H, —CH₂N₃), 7.50 (d, 2H, J=5Hz), 7.9 (m, 4H), and 8.03 (d, 2H, J=10 Hz)

3,3′-isomer—4.54 (s, 2H, —CH₂N₃), 7.4 (d, 1H, J=10 Hz), 7.5-7.7 (m, 4H),7.92 (ABq, 2H) and 8.19 (s, 1H).

4. Reduction of the Azides to the Amine

-   -   This was carried out on the solid phase and the amine was never        isolated. The azidocarboxylic acid was loaded on the resin using        the standard peptide coupling protocols. After washing, the        resin containing the azide was shaken with 20 equivalents of        triphenylphosphine in THF/water (95:5) for 24 h. The solution        was drained under a positive pressure of nitrogen and then        washed with the standard washing procedure. The resulting amine        was employed in the next coupling.

5.(3β,5β,7α,12α)-3-[{(9H-Flouren-9ylmethoxy)amino]acetyl}amino-7,12-dihydroxycholan-24-oicacid

-   -   Tributylamine (3.2 mL); 13.5 mmol) was added dropwise to a        solution of Fmoc-glycine (4.0 g, 13.5 mmol) in THF (80 mL)        stirred at 0° C.

Isobutylchloroformate (1.7 mL; 13.5 mmol) was subsequently added and,after 10 min, a suspension of tributylamine (2.6 mL; 11.2 mmol) and(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid (4.5 g; 11.2mmol) in DMF (80 mL) was added dropwise, over 1 h, into the cooledsolution. The mixture was allowed to warm up to ambient temperature andafter 6 h, the solution was concentrated to 120 mL, then water (180 mL)and 1N HCl (30 mL) were added (final pH 1.5). The precipitated solid wasfiltered, washed with water (2×100 mL), vacuum dried and purified byflash chromatography. Elution with chloroform/methanol (8:2) yielded theproduct as a colorless solid.

Yield: 1.9 g (25%). TLC: R_(f) 0.30 (CHCl₃/MeOH/NH₄OH—6:3:1).

In Vitro and In Vivo Testing of Compounds Example X In Vitro BindingAssay for GRP Receptors in PC3 Cell Lines—FIGS. 9A-B

To identify potential lead compounds, an in vitro assay that identifiescompounds with high affinity for GRP-R was used. Since the PC3 cellline, derived from human prostate cancer, is known to exhibit highexpression of GRP-R on the cell surface, a radio ligand binding assay ina 96-well plate format was developed and validated to measure thebinding of ¹²⁵I-BBN to GRP-R positive PC3 cells and the ability of thecompounds of the invention to inhibit this binding. This assay was usedto measure the IC₅₀ for RP527 ligand, L134 ligand (controls) andcompounds of the invention which inhibit the binding of ¹²⁵I-BBN toGRP-R. (RP527=N,N-dimethylglycine-Ser-Cys(Acm)-Gly-5-aminopentanoicacid-BBN (7-14) [SEQ. ID. NO: 1], which has MS=1442.6 and IC50—0.84).Van de Wiele C, Dumont F et al., Technetium-99m RP527, a GRP analoguefor visualization of GRP receptor-expressing malignancies: a feasibilitystudy. Eur. J. Nucl. Med., (2000) 27; 1694-1699.;L134=DO3A-monoamide-8-amino-octanoic acid-BBN (7-14) [SEQ. ID. NO: 1],which has MS=1467.0.

-   -   The Radioligand Binding Plate Assay was validated for BBN and        BBN analogues (including commercially available BBN and L1) and        also using ^(99m)Tc RP527 as the radioligand.

A. Materials and Method:

1. Cell Culture:

-   -   PC3 (human prostate cancer cell line) were obtained from the        American Type Culture Collection and cultured in RPMI 1640 in        tissue culture flasks (Corning). This growth medium was        supplemented with 10% heat inactivated FBS (Hyclone,        SH30070.03), 10 mM HEPES (GibcoBRL, 15630-080), and        antibiotic/antimycotic (GibcoBRL, 15240-062) for a final        concentration of penicillin-streptomycin (100 units/mL), and        fungizone (0.25 μg/mL). All cultures were maintained in a        humidified atmosphere containing 5% CO₂/95% air at 37° C., and        passaged routinely using 0.05% trypsin/EDTA (GibcoBRL 25300-054)        where indicated. Cells for experiments were plated at a        concentration of 2.0×10⁴/well either in 96-well white/clear        bottom microtiter plates (Falcon Optilux-I) or 96 well        black/clear collagen I cellware plates (Beckton Dickinson        Biocoat). Plates were used for binding studies on day 1 or 2        post-plating.

2. Binding Buffer:

-   -   RPMI 1640 supplemented with 20 mM HEPES, 0.1% BSA (w/v), 0.5 mM        PMSF (AEBSF), bacitracin (50 mg/500 ml), pH 7.4. ¹²⁵I-BBN        (carrier free, 2200 Ci/mmole) was obtained from Perkin-Elmer.

B. Competition Assay with ¹²⁵I-BBN for GRP-R in PC3 cells:

-   -   A 96-well plate assay was used to determine the IC₅₀ of various        compounds of the invention to inhibit binding of ¹²⁵I-BBN to        human GRP-R. The following general procedure was followed:

All compounds tested were dissolved in binding buffer and appropriatedilutions were also done in binding buffer. PC3 cells (human prostatecancer cell line) for assay were plated at a concentration of2.0×10⁴/well either in 96-well white/clear bottomed microtiter plates(Falcon Optilux-I) or 96 well black/clear collagen I cellware plates(Beckton Dickinson Biocoat). Plates were used for binding studies on day1 or 2 post-plating. The plates were checked for confluency (>90%confluent) prior to assay. For the assay, RP527 or L134 ligand,(controls), or compounds of the invention at concentrations ranging from1.25×10⁻⁹M to 5×10⁻⁹ M, was co-incubated with ¹²⁵I-BBN (25,000cpm/well). These studies were conducted with an assay volume of 75 μlper well. Triplicate wells were used for each data point. After theaddition of the appropriate solutions, plates were incubated for 1 h at4° C. to prevent internalization of the ligand-receptor complex.Incubation was ended by the addition of 200 μl of ice-cold incubationbuffer. Plates were washed 5 times and blotted dry. Radioactivity wasdetected using either the LKB CompuGamma counter or a microplatescintillation counter.

Competition binding curves for RP527 (control) and L70, a compound ofthe invention can be found in FIG. 9A-B These data show that the IC50 ofthe RP527 control is 2.5 nM and that of L70, a compound of thisinvention is 5 nM. The IC50 of the L134 control was 5 nM. IC50 valuesfor those compounds of the invention tested can be found in tables 1-3and show that they are comparable to that of the controls and thus wouldbe expected to have sufficient affinity for the receptor to allow uptakeby receptor bearing cells in vivo.

C. Internalization & Efflux Assay:

-   -   These studies were conducted in a 96-well plate. After washing        to remove serum proteins, PC3 cells were incubated with        ¹²⁵I-BBN, ¹⁷⁷Lu-L134 or radiolabelled compounds of this        invention for 40 min, at 37° C. Incubations were stopped by the        addition of 200 μl of ice-cold binding buffer. Plates were        washed twice with binding buffer. To remove surface-bound        radioligand, the cells were incubated with 0.2M acetic acid (in        saline), pH 2.8 for 2 min. Plates were centrifuged and the acid        wash media were collected to determine the amount of        radioactivity which was not internalized. The cells were        collected to determine the amount of internalized ¹²⁵I-BBN, and        all samples were analyzed in the gamma counter. Data for the        internalization assay was normalized by comparing counts        obtained at the various time points with the counts obtained at        the final time point (T40 min). For the efflux studies, after        loading the PC3 cells with ¹²⁵I-BBN or radiolabelled compounds        of the invention for 40 min at 37° C., the unbound material was        washed off, and the % of internalization was determined as        above. The cells were then resuspended in binding buffer at        37° C. for up to 3 h At 0.5, 1, 2, or 3 h. the amount remaining        internalized relative to the initial loading level was        determined as above and used to calculate the percent efflux        recorded in Table 4.

TABLE 4 Internalisation and efflux ofI-BBN and the Lu-177 complexes ofL134 (control) and compounds of this invention I-BBN L134 L63 L64 L70Internalisation (40 minutes) 59 89 64 69 70 Efflux (2 h) 35 28 0 20 12These data show that the compounds of this invention are internalizedand retained by the PC3 cells to a similar extent to the controls.

Example XI Preparation of Tc-Labeled GRP Compounds

Peptide solutions of compounds of the invention identified in Table 5were prepared at a concentration of 1 mg/mL in 0.1% aqueous TFA. Astannous chloride solution was prepared by dissolving SnCl₂.2H₂O (20mg/mL) in 1 N HCl. Stannous gluconate solutions containing 20 μg ofSnCl₂.2H₂O/100 μL were prepared by adding an aliquot of the SnCl₂solution (10 μL) to a sodium gluconate solution prepared by dissolving13 mg of sodium gluconate in water. A hydroxypropyl gamma cyclodextrin[HP-γ-CD] solution was prepared by dissolving 50 mg of HP-γ-CD in 1 mLof water.

The ^(99m)Tc labeled compounds identified below were prepared by mixing20 μL of solution of the unlabeled compounds (20 μg), 50 μL of HP-γ-CDsolution, 100 μL of Sn-gluconate solution and 20 to 50 μL of ^(99m)Tcpertechnetate (5 to 8 mCi, Syncor). The final volume was around 200 μLand final pH was 4.5-5. The reaction mixture was heated at 100° C. for15 to 20 min. and then analyzed by reversed phase HPLC to determineradiochemical purity (RCP). The desired product peaks were isolated byHPLC, collected into a stabilizing buffer containing 5 mg/mL ascorbicacid, 16 mg/mL HP-γ-CD and 50 mM phosphate buffer, pH 4.5, andconcentrated using a speed vacuum to remove acetonitrile. The HPLCsystem used for analysis and purification was as follows: C18 Vydaccolumn, 4.6×250 mm, aqueous phase: 0.1% TFA in water, organic phase:0.085% TFA in acetonitrile. Flow rate: 1 mL/min. Isocratic elution at20%-25% acetonitrile/0.085% TFA was used, depending on the nature ofindividual peptide.

Labeling results are summarized in Table 5.

TABLE 5 HPLC RCP⁴ (%) retention Initial immediately time RCP³ followingCompound¹ Sequence² (min) (%) purification L2 -RJQWAVGHLM 5.47 89.9 95.6L4 -SJQWAVGHLM 5.92 65 97 L8 -JKQWAVGHLM 6.72 86 94 L1 -KJQWAVGHLM 5.4388.2 92.6 L9 -JRQWAVGHLM 7.28 91.7 96.2 L7 -aJQWAVGHLM 8.47 88.6 95.9n.d. = not detected ¹All compounds were conjugated with anN,N′-dimethylglycine-Ser-Cys-Gly metal chelator. The Acm protected formof the ligand was used. Hence, the ligand used to prepare the 99mTccomplex of L2 was N,N′-dimethylglycine-Ser-Cys(Acm)-Gly-RJQWAVGHLM. TheAcm group was removed during chelation to Tc. ²In the Sequence “J”refers to 8-amino-3,6-dioxaoctanoic acid and “a” refers to D-alanine³Initial RCP measurement taken immediately after heating and prior toHPL purification. ⁴RCP determined following HPLC isolation andacetonitrile removal via speed vacuum

Example XII Preparation of ¹⁷⁷Lu-L64 for Cell Binding andBiodistribution Studies

This compound was synthesized by incubating 10 μL64 ligand (10 μL of a 1mg/mL solution in water), 100 μL ammonium acetate buffer (0.2M, pH 5.2)and 1-2 mCi of ¹⁷⁷LuCl₃ in 0.05N HCl (MURR) at 90° C. for 15 min. Free¹⁷⁷Lu was scavenged by adding 20 μL of a 1% Na₂EDTA.2H₂O (Aldrich)solution in water. The resulting radiochemical purity (RCP) was ˜95%.The radiolabeled product was separated from unlabeled ligand and otherimpurities by HPLC, using a YMC Basic C8 column [4.6×150 mm], a columntemperature of 30° C. and a flow rate of 1 mL/min, with a gradient of68% A/32% B to 66% A/34% B over 30 min., where A is citrate buffer(0.02M, pH 3.0), and B is 80% CH₃CN/20% CH₃OH. The isolated compound hadan RCP of 100% and an HPLC retention time of 23.4 minutes.

Samples for biodistribution and cell binding studies were prepared bycollecting the desired HPLC peak into 1000 μL of citrate buffer (0.05 M,pH 5.3, containing 1% ascorbic acid, and 0.1% HSA). The organic eluentin the collected eluate was removed by centrifugal concentration for 30min. For cell binding studies, the purified sample was diluted withcell-binding media to a concentration of 1.5 μCi/mL within 30 minutes ofthe in vitro study. For biodistribution studies, the sample was dilutedwith citrate buffer (0.05 M, pH 5.3, containing 1% sodium ascorbic acidand 0.1% HSA) to a final concentration of 50 μCi/mL within 30 minutes ofthe in vivo study.

Example XIII Preparation of ¹⁷⁷Lu-L64 for Radiotherapy Studies

This compound was synthesized by incubating 70 μL64 ligand (70 μL of a 1mg/mL solution in water), 200 μL ammonium acetate buffer (0.2M, pH 5.2)and 30-40 mCi of ¹⁷⁷LuCl₃ in 0.05N HCl (MURR) at 85° C. for 10 min.After cooling to room temperature, free ¹⁷⁷Lu was scavenged by adding 20μL of a 2% Na₂EDTA.2H₂O (Aldrich) solution in water. The resultingradiochemical purity (RCP) was 95%. The radiolabeled product wasseparated from unlabeled ligand and other impurities by HPLC, using a300VHP Anion Exchange column (7.5×50 mm) (Vydac) that was sequentiallyeluted at a flow rate of 1 mL/min with water, 50% acetonitrile/water andthen 1 g/L aqueous ammonium acetate solution. The desired compound waseluted from the column with 50% CH₃CN and mixed with 1 mL of citratebuffer (0.05 M, pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and 0.9%(v:v) benzyl alcohol. The organic part of the isolated fraction wasremoved by spin vacuum for 40 min, and the concentrated solution (˜20-25mCi) was adjusted within 30 minutes of the in vivo study to aconcentration of 7.5 mCi/mL using citrate buffer (0.05 M, pH 5.3)containing 5% ascorbic acid, 0.2% HSA, and 0.9% (v:v) benzyl alcohol.The resulting compound had an RCP of >95%.

Example XIV Preparation of ¹¹¹In-L64

This compound was synthesized by incubating 10 μL64 ligand (5 μL of a 2mg/mL solution in 0.01 N HCl), 60 μL ethanol, 1.12 mCi of ¹¹¹InCl₃ in0.05N HCl (80 μL) and 155 μL sodium acetate buffer (0.5M, pH 4.5) at 85°C. for 30 min. Free ¹¹¹In was scavenged by adding 20 μL of a 1%Na₂EDTA.2H₂O (Aldrich) solution in water. The resulting radiochemicalpurity (RCP) was 87%. The radiolabeled product was separated fromunlabeled ligand and other impurities by HPLC, using a Vydac C18 column,[4,6×250 mm], a column temperature of 50° C. and a flow rate of 1.5mL/min. with a gradient of 75% A/25% B to 65% A/35% B over 20 min whereA is 0.1% TFA in water, B is 0.085% TFA in acetonitrile. With thissystem, the retention time for ¹¹¹In-L64 is 15.7 min. The isolatedcompound had an RCP of 96.7%.

Example XV Preparation of ¹⁷⁷Lu-L134 (Control)

A stock solution of peptide was prepared by dissolving L134 ligand(prepared as described in US Application Publication No. 2002/0054855and WO 02/87637, both incorporated by reference) in 0.01 N HCl to aconcentration of 1 mg/mL ¹⁷⁷Lu-L134 was prepared by mixing the followingreagents in the order shown.

0.2 M NH₄OAc, pH 6.8 100 μl Peptide stock, 1 mg/mL, in 0.01 N HCl 5 μl¹⁷⁷LuCl₃ (MURR) in 0.05M HCl 1.2 μl (1.4 mCi)The reaction mixture was incubated at 85° C. for 10 min. After coolingdown to room temperature in a water bath, 20 μl of a 1% EDTA solutionand 20 μl of EtOH were added. The compound was analyzed by HPLC using aC18 column (VYDAC Cat #218TP54) that was eluted at flow rate of 1 mL/minwith a gradient of 21 to 25% B over 20 min, where A is 0.1% TFA/H₂O andB is 0.1% TFA/CH₃CN). ¹⁷⁷Lu-L134 was formed in 97.1% yield (RCP) and hada retention time of 16.1 min on this system.

Example XVI Preparation of ¹⁷⁷Lu-L63

This compound was prepared as described for ¹⁷⁷Lu-L134. The compound wasanalyzed by HPLC using a C18 column (VYDAC Cat #218TP54) that was elutedat flow rate of 1 mL/min with a gradient of 30-34% B over 20 min (wheresolvent is A. 0.1% TFA/H₂O and B is 01% TFA/CH₃CN). The ¹⁷⁷Lu-L63 thatformed had an RCP of 97.8% and a retention time of ˜14.2 min on thissystem.

Example XVII Preparation of ¹⁷⁷Lu-L70 for Cell Binding andBiodistribution Studies

This compound was prepared following the procedures described above, butsubstituting L70 (the ligand of Example II). Purification was performedusing a YMC Basic C8 column (4.6×150 mm), a column temperature of 30° C.and a flow rate of 1 mL/min. with a gradient of 80% A/20% B to 75% A/25%B over 40 min., where A is citrate buffer (0.02M, pH 4.5), and B is 80%CH₃CN/20% CH₃OH. The isolated compound had an RCP of 100% and an HPLCretention time of 25.4 min.

Example XVIII Preparation of ¹⁷⁷Lu-L70 for Radiotherapy Studies

This compound was prepared as described above for L64.

Example XIX Preparation of ¹¹¹In-L70 for Cell Binding andBiodistribution Studies

This compound was synthesized by incubating 10 μL70 ligand (10 μL of a 1mg/mL solution in 0.01 N HCl), 180 μL ammonium acetate buffer (0.2M, pH5.3), 1.1 mCi of ¹¹¹InCl₃ in 0.05N HCl (61 μL, Mallinckrodt) and 50 μLof saline at 85° C. for 30 min. Free ¹¹¹In was scavenged by adding 20 μLof a 1% Na₂EDTA.2H₂O (Aldrich) solution in water. The resultingradiochemical purity (RCP) was 86%. The radiolabeled product wasseparated from unlabeled ligand and other impurities by HPLC, using aWaters XTerra C18 cartridge linked to a Vydac strong anion exchangecolumn [7.5×50 mm], a column temperature of 30° C. and a flow rate of 1mL/min. with the gradient listed in the Table below, where A is 0.1 mMNaOH in water, pH 10.0, B is 1 g/L ammonium acetate in water, pH 6.7 andC is acetonitrile. With this system, the retention time for ¹¹¹In-L70 is15 min while the retention time for L70 ligand is 27 to 28 min. Theisolated compound had an RCP of 96%.

Samples for biodistribution and cell binding studies were prepared bycollecting the desired HPLC peak into 500 μL of citrate buffer (0.05 M,pH 5.3, containing 5% ascorbic acid, 1 mg/mL L-methionine and 0.2% HSA).The organic part of the collection was removed by spin vacuum for 30min. For cell binding studies, the purified, concentrated sample wasused within 30 minutes of the in vitro study. For biodistributionstudies, the sample was diluted with citrate buffer (0.05 M, pH 5.3,containing 5% sodium ascorbic acid and 0.2% HSA) to a finalconcentration of 10 μCi/mL within 30 minutes of the in vivo study.

Time, min A B C  0-10 100% 10-11 100-50% 0-50% 11-21  50% 50% 21-2250-0% 0-50% 50% 22-32 50% 50%

Example XX In Vivo Pharmacokinetic Studies

A. Tracer Dose Biodistribution:

-   -   Low dose pharmacokinetic studies (e.g., biodistribution studies)        were performed using the below-identified compounds of the        invention in xenografted, PC3 tumor-bearing nude mice        ([Ncr]-Foxn1<nu>). In all studies, mice were administered 100 μL        of ¹⁷⁷Lu-labelled test compound at 200 μCi/kg, i.v., with a        residence time of 1 and 24 h per group (n=3-4). Tissues were        analyzed in an LKB 1282 CompuGamma counter with appropriate        standards.

TABLE 6 Pharmacokinetic comparison at 1 and 24 h in PC3 tumor-bearingnude mice (200 μCi/kg; values as % ID/g) of Lu-177 labelled compounds ofthis invention compared to control L134 control L63 L64 L70 Tissue 1 hr24 hr 1 hr 24 hr 1 hr 24 hr 1 hr 24 hr Blood 0.44 0.03 7.54 0.05 1.870.02 0.33 0.03 Liver 0.38 0.04 12.15 0.20 2.89 0.21 0.77 0.10 Kidneys7.65 1.03 7.22 0.84 10.95 1.45 6.01 2.31 Tumor 3.66 1.52 9.49 2.27 9.833.60 6.42 3.50 Pancreas 28.60 1.01 54.04 1.62 77.78 6.56 42.34 40.24Whereas the distribution of radioactivity in the blood, liver andkidneys after injection of L64 and L70 is similar to that of the controlcompound, L134, the uptake in the tumor is much higher at 1 and 24 h forboth L64 and L70. L63 also shows high tumour uptake although withincreased blood and liver values at early times. Uptake in the mousepancreas, a normal organ known to have GRP receptors is much higher forL64, L70 and L63 than for L134.

Example XXI Radiotherapy Studies

A. Short Term Efficacy Studies:

-   -   Radiotherapy studies were performed using the PC3 tumor-bearing        nude mouse model. Lu-177 labelled compounds of the invention        L64, L70, L63 and the treatment control compound L134 were        compared to an untreated control group. (n=12 for each treatment        group and n=36 for the untreated control group). For the first        study, mice were administered 100 μL of ¹⁷⁷Lu-labelled compound        of the invention at 30 mCi/kg, i.v, or s.c. under sterile        conditions. The subjects were housed in a barrier environment        for up to 30 days. Body weight and tumor size (by caliper        measurement) were collected on each subject 3 times per week for        the duration of the study. Criteria for early termination        included: death; loss of total body weight (TBW) equal to or        greater than 20%; tumor size equal to or greater than 2 cm³.        Results are displayed in FIG. 10A. These results show that        animals treated with L70, L64 or L63 have increased survival        over the control animals given no treatment and over those        animals given the same dose of L134.    -   A repeat study was performed with L64 and L70 using the same        dose as before but using more animals per compound (n=46) and        following them for longer. The results of the repeat study are        displayed in FIG. 10B. Relative to the same controls as before        (n=36), both L64 and L70 treatment give significantly increased        survival (p<0.0001) with L70 being better than L64 (p0.079).

Example XXII Alternative Preparation of L64 and L70 Using SegmentCoupling

Compounds L64 and L70 can be prepared employing the collection ofintermediates generally represented by A-D (FIG. 14), which themselvesare prepared by standard methods known in the art of solid and solutionphase peptide synthesis (Synthetic Peptides—A User's Guide 1992, Grant,G., Ed. WH. Freeman Co., NY, Chap 3 and Chap 4 pp 77-258; Chan, W. C.and White, P. D. Basic Procedures in Fmoc Solid Phase PeptideSynthesis—A Practical Approach 2002, Chan, W. C. and White, P. D. EdsOxford University Press, New York, Chap. 3 pp 41-76; Barlos, K andGatos, G. Convergent Peptide Synthesis in Fmoc Solid Phase PeptideSynthesis—A Practical Approach 2002, Chan, W. C. and White, P. D. EdsOxford University Press, New York, Chap. 9 pp 216-228.) which areincorporated herein by reference.

These methods include Aloc, Boc, Fmoc or benzyloxycarbonyl-based peptidesynthesis strategies or judiciously chosen combinations of those methodson solid phase or in solution. The intermediates to be employed for agiven step are chosen based on the selection of appropriate protectinggroups for each position in the molecule, which may be selected from thelist of groups shown in FIG. 1. Those of ordinary skill in the art willalso understand that intermediates, compatible with peptide synthesismethodology, comprised of alternative protecting groups can also beemployed and that the listed options for protecting groups shown aboveserves as illustrative and not inclusive, and that such alternatives arewell known in the art.

This is amply illustrated in FIG. 15 which outlines the approach.Substitution of the intermediate C2 in place of C1 shown in thesynthesis of L64, provides L70 when the same synthetic strategies areapplied.

We claim:
 1. A composition comprising a compound of the general formula:M-N-O-P-G wherein M is a metal chelator, optionally complexed with aradionuclide; N is absent, an alpha amino acid, a substituted bile acidor other linking group; O is an alpha amino acid or a substituted bileacid; and P is absent, an alpha amino acid, a substituted bile acid orother linking group; and G is a GRP receptor targeting peptide, andwherein at least one of N, O or P is a substituted bile acid.
 2. Thecomposition of claim 1, wherein G is an agonist.
 3. The composition ofclaim 1, wherein the substituted bile acid is selected from the groupconsisting of: 3β-amino-3-deoxycholic acid; (3β,5β)-3-aminocholan-24-oicacid; (3β,5β,12α)-3-amino-12-hydroxycholan-24-oic acid;(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid;Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholicacid); (3β,5β,7α,12α)-3-amino-7-hydroxy-12-oxocholan-24-oic acid; and(3β,5β,7α)-3-amino-7-hydroxycholan-24-oic acid.
 4. The composition ofclaim 1, wherein M is selected from the group consisting of: DTPA, DOTA,DO3A, HP-DO3A, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA,LICAM, MECAM, and CMDOTA.
 5. The composition of claim 4, wherein M isEHPG.
 6. The composition of claim 1, wherein M is selected from thegroup consisting of 5-Cl-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and5-sec-Bu-EHPG.
 7. The composition of claim 4, wherein M isbenzodiethylenetriamine pentaacetic acid (benzo-DTPA).
 8. Thecomposition of claim 1, wherein M is selected from the group consistingof dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA.
 9. The composition of claim 4, wherein M is HBED.
 10. Thecomposition of claim 4, wherein M is selected from the group consistingof benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, benzo-TETA, benzo-DOTMA,and benzo-TETMA.
 11. The composition of claim 4, wherein M is selectedfrom the group consisting of 1,3-propylenediaminetetraacetic acid(PDTA); triethylenetetraaminehexaacetic acid (TTHA);1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). 12.The composition of claim 1, wherein M is selected from the groupconsisting of: N,N-dimethylGly-Ser-Cys; N,N-dimethylGly-Thr-Cys;N,N-diethylGly-Ser-Cys; and N,N-dibenzylGly-Ser-Cys.
 13. The compositionof claim 1, wherein M is selected from the group consisting of:N,N-dimethylGly-Ser-Cys-Gly; N,N-dimethylGly-Thr-Cys-Gly;N,N-diethylGly-Ser-Cys-Gly; and N,N-dibenzylGly-Ser-Cys-Gly.
 14. Acomposition of claim 1, selected from the group consisting of:DO3A-monoamide-Gly-(3β,5β)-3-aminocholan-24-oic acid-BBN(7-14) whereinthe BBN(7-14) sequence is SEQ. ID NO: 1;DO3A-monoamide-Gly-(3β,5β,12α)-3-amino-12-hydroxycholan-24-oicacid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;DO3A-monoamide-Gly-(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;DO3A-monoamide-Gly-Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholicacid)-Arg-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1;(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-3,6,9-trioxaundecane-1,11-dicarbonylLys(DO3A-monoamide-Gly)-Arg-BBN(7-14) wherein the BBN(7-14) sequence isSEQ. ID NO: 1;DO3A-monoamide-Gly-(3β,5β,7α,12β)-3-amino-12-oxocholan-24-oicacid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1; andDO3A-monoamide-1-amino-3,6-dioxaoctanoicacid-(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oic acid-BBN(7-14)wherein the BBN(7-14) sequence is SEQ. ID NO:
 1. 15. A method of imagingcomprising the steps of: administering to a patient a diagnostic imagingagent comprising the composition of anyone of claim 1 or 14, complexedwith a diagnostic radionuclide, and imaging GRP-R expressing tissue insaid patient.
 16. A method for preparing a diagnostic imaging agentcomprising the step of adding to an injectable medium a substancecomprising the composition of anyone of claim 1 or
 14. 17. A method oftreating tumors in a patient in need of radiotherapy comprising the stepof administering to a patient a radiotherapeutic agent comprising thecomposition of anyone of claim 1 or 14, complexed with a therapeuticradionuclide.
 18. A method of preparing a radiotherapeutic agentcomprising the step of adding to an injectable medium a substancecomprising the composition of anyone of claim 1 or
 14. 19. A compositioncomprisingDO3A-monoamide-Gly(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN (7-14) wherein the BBN(7-14) sequence is SEQ ID NO:
 1. 20. Amethod of imaging comprising the steps of: administering to a patient adiagnostic imaging agent comprisingDO3A-monoamide-Gly-(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN(7-14) complexed with a diagnostic radionuclide, wherein theBBN(7-14) sequence is SEQ ID NO: 1, and imaging GRP-R expressing tissuein said patient.
 21. A method for preparing a diagnostic imaging agentcomprising the step of adding to an injectable medium a compositioncomprising DO3A-monoamide-Gly-4-aminobenzoic acid-BBN(7-14) wherein theBBN(7-14) sequence is SEQ ID NO:
 1. 22. A method of treating a tumor ina patient in need of radiotherapy comprising the step of administeringto a patient a radiotherapeutic agent comprising the composition ofclaim 18 complexed with a therapeutic radionuclide.
 23. A method ofsynthesizingDO3A-monoamide-Gly(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN (7-14) wherein the BBN(7-14) sequence is SEQ ID NO: 1comprising the steps of: (a) shaking a solution in a solid phase peptidesynthesis vessel, said solution comprising a resin and at least onepeptide building ingredient, (b) flushing said solution, and (c) washingsaid resin with DMA, wherein said at least one peptide buildingingredient includes DMAC morpholine,(3β,5β,7α,12α)-3-[[(9H-fluoren-9-ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oicacid, HOBt, DIC, HATU or mixtures thereof, and wherein each of steps(a), (b) and (c) are repeated untilDO3A-monoamide-Gly(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN (7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1 isobtained.
 24. A method for labelingDO3A-monoamide-Gly(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN (7-14) wherein the BBN(7-14) sequence is SEQ. ID NO: 1comprising the steps of incubating a first solution comprisingDO3A-monoamide-Gly-(3β,5β,7α,12α)-3-amino-7,12-dihydroxycholan-24-oicacid-BBN(7-14) wherein the BBN(7-14) sequence is SEQ ID NO: 1, ammoniumacetate, a radioactive metal precursor selected from the groupconsisting of ¹⁷⁷LuCl₃ or ¹¹¹InCl₃ in HCl, and adding to said firstsolution a second solution comprising Na₂EDTA.2H₂O and water to obtain aradiochemical purity greater than 95%.